An improved approach for transformation of plant cells by microinjection: molecular and genetic analysis

A new culture method for the injection of tobacco mesophyll protoplasts has been established. The protoplasts are embedded in a thin layer of alginate and are nourished from the medium in the underlying basislayer. In the alginate layer the protoplasts regenerate to calli at a frequency of up to 80%. Embedded protoplasts can be selected either with 50 mg l⁻¹ kanamycin or 5 mg l⁻¹ paromomycin. Single resistant cells can be recovered from about 10 000 sensitive cells in one alginate layer. Injection of theneo gene (coding for neomycin phosphotransferase II) into protoplast derived single cells in the alginate layer results in kanamycin resistant colonies that can be regenerated to mature plants. These plants express the neomycin phosphotransferase as shown by enzyme activity assay. The integration of the transgene into the plant genome could be proved by Southern hybridization to high molecular weight DNA. With this culture method 100 cells can be injected per hour. Transformation frequencies range from 2 to 20%. In crossing experiments, it was shown that the foreign gene is transmitted to the next generation in a Mendelian fashion.     

The human recombinant c-kit receptor ligand, rhSCF, induces mediator release from human cutaneous mast cells and enhances IgE-dependent mediator release from both skin mast cells and peripheral blood basophils.

The gene product of the steel locus of the mouse represents a growth factor for murine mast cells and a ligand for the c-kit proto-oncogene receptor, a member of the tyrosine kinase receptor class of oncogenes (for review, see O. N. Witte. 1990. Cell 63:5). We have studied the effect of the human recombinant c-kit receptor ligand stem cell factor (rhSCF) on the release of inflammatory mediators from human skin mast cells and peripheral blood basophils and compared its activity to that of rhIL-3, rhSCF (1 ng/ml to 1 microgram/ml) activated the release of histamine and PGD2 from mast cells isolated from human skin. Analysis by digital video microscopy indicated that purified human skin mast cells (84 +/- 5% pure) responded to rhSCF (0.1 to 1 microgram/ml) challenge with a rapid, sustained rise in intracellular Ca2+ levels that was accompanied by secretion of histamine. A brief preincubation (10 min) of mast cells with rhSCF (0.1 pg/ml to 1 ng/ml) significantly enhanced (100 +/- 35%) the release of histamine induced by anti-IgE (3 micrograms/ml), but was much less effective on IgE-mediated release of PGD2. In contrast, a short term incubation with rhSCF did not potentiate the secretion of histamine activated by substance P (5 microM). A 24-h incubation of mast cells with rhSCF did not affect the release of mediators induced by anti-IgE (3 micrograms/ml), probably due to receptor desensitization, rhSCF (1 ng/ml to 3 micrograms/ml) neither caused release of histamine or leukotriene C4 (LTC4) release from leukocytes of 14 donors, nor induced a rise in intracellular Ca2+ levels in purified (greater than 70%) basophils. Brief preincubation (10 min) of leukocytes with rhSCF (1 ng/ml to 3 micrograms/ml) caused an enhancement (69 +/- 11%) of anti-IgE-induced release of histamine that was significant at concentrations as low as 3 ng/ml (p less than 0.05), whereas it appeared less effective in potentiating IgE-mediated LTC4 release. In contrast, a prolonged incubation (24 h) with rhSCF (0.1 pg/ml to 100 ng/ml) did not enhance the release of histamine or LTC4 induced by anti-IgE (0.1 microgram/ml), whereas rhIL-3 (3 ng/ml) significantly potentiated the release of both mediators.(ABSTRACT TRUNCATED AT 400 WORDS)     

Retinoic acid-induced cartilage degradation is caused by cartilage cells

Summary Cartilage cubes, prepared from the proximal epiphyses of neonatal rat humeri and consisting of cartilage tissue only, were cultured in the presence of retinoic acid. The retinoid induced the loss of metachromatic staining with toluidine blue, which correlates with the loss of proteoglycan, followed by tissue degradation processes resulting in a distinct reduction of the cartilage mass. Histologically, fibroblast-like cells appeared within chondrones, indicating a transformation of chondroblasts. Focal tissue degradation was observed after only 2 days. Electron microscopically, the clustered cells within the zone of tissue degradation were rich in various lysosomal structures indicating their lytic activity. Cycloheximide and EDTA completely blocked the retinoic acid effects suggesting that protein synthesis was required and that metalloproteinases may be involved in the degradation processes. In conclusion, with the new test system described here we demonstrated that cartilage cells themselves performed the tissue degradation induced by retinoic acid.     

Evaluation of 3-hydroxy-3-methylglutaryl-CoA reductase activity by multiple-selected ion monitoring

A new method for the evaluation of 3-hydroxy-3-methylglutaryl-CoA reductase activity is described, based on the multiple-selected ion monitoring of the amount of mevalonate formed in incubations of 3-hydroxy-3-methylglutaryl-CoA with microsomal proteins. Analysis is carried out on crude extracts using deuterated mevalonic acid lactone as internal standard. The sensitivity of the technique allows the quantitative evaluation of mevalonate in microassays (100 μg microsomal protein) of the enzyme activity at the minimum value of the diurnal rhythm.     

PGF2 alpha, thromboxane B2 and HETE levels in gerbil brain cortex after ligation of common carotid arteries and decapitation.

The effects of ligation of both common carotid arteries in the gerbil on the levels of PGF2 alpha, TXB2, HETE and of energy metabolites in brain cortex, have been investigated. Also, in the same experimental conditions the changes of cyclic AMP in brain cortex, cerebellum, striatum and hippocampus have been monitored. ATP, glycogen, glucose and phosphocreatine decrease whereas, lactate and cyclic AMP are enhanced in the ischemic brain, as previously reported. In contrast, levels of arachidonic acid metabolites are not modified. During ischemia following decapitation, instead, PGF2 alpha, and TXB2, show considerable increase.     

Differential effects of probenecid on the levels of endogenous PGF2 alpha and TXB2 in brain cortex

Probenecid in single or repeated doses does not modify levels of PGF2 alpha and TXB2 in rat brain cortex. After administration of subconvulsant dose of pentamethylene tetrazole (PMT) PGF2 alpha increases sharply and rapidly declines subsequently, whereas the elevation of TXB2 is smaller but of longer duration. After probenecid pretreatment PGF2 alpha levels do not decline up to 30 minutes after the initial peak and are still elevated after 60 minutes. Levels of TXB2 tend to be reduced after pretreatment. Differences in transport process or in biosynthetic compartments for these arachidonic acid (AA) metabolites may account for the observed data.     

Differential effects of probenecid on the levels of endogenous PGF2α and TXB2 in brain cortex

Probenecid in single or repeated doses does not modify levels of PGF_2α and TXB₂ in rat brain cortex. After administration of subconvulsant dose of pentamethylene tetrazone (PMT) PGF_2α increases sharply and rapidly declines subsequently, whereas the elevation of TXB₂ is smaller but of longer duration. After probenecid pretreatment PGF_2α levels do not decline up to 30 minutes after the initial peak and are still elevated after 60 minutes. Levels of TXB₂ tend to be reduced after pretreatment. Differences in transport process or in biosynthetic compartments for these arachidonic acid (AA) metabolites may account for the observed data.     

A mass fragmentographic method for the quantitative evaluation of brain prostaglandin biosynthesis.

A method for the evaluation of PGF-2alpha and PGE-2 biosynthesis in rat cerebral cortex is described. Tissue slices were incubated without any added precursor for different lengths of time. The analytical procedure involves prostaglandin extraction, purification and quantitative determination by mass fragmentography. Significant amounts of both prostaglandins were synthesized. The biosynthesis reached a plateau after 30 minutes and the ratio of PGF-2alpha to PGE-2 was approximately 3.