Freeze-preservation of rice cells: A physiological study of freeze-thawed cells

Rice ( Oryza sativa L.) cells returning to in vitro culture after preservation at superlow temperature in liquid nitrogen are characterized by a number of physiological alterations. These include: reduction in respiration and glucose uptake, loss of intracellular potassium, decrease in the cellular level of key metabolites (ATP, glucose-6-phosphate and pyruvate) and fragility of protoplasts following the action of cell wall-degrading enzymes.
Nevertheless, cell growth resumes after a short lag phase (2–4 days) with an actual 70–100% cell survival, thus indicating that the observed damage is not lethal and can be repaired in a short time.     

Inhibition of elongation and H+ and K+ transport by the phosphodiesterase inhibitor aminophylline in plant tissue

Aminophylline, an inhibitor of cyclic nucleotide phosphodiesterase (EC 3.1.4.17), inhibits elongation and correlated H⁺ and K⁺ transport in embryos of Haplopappus gracilis and in pea internode segments. Moreover, the drug strongly inhibits the stimulation of these processes by fusicoccin and indole-3-acetic acid and reduces passive permeability of the membrane. The possible mechanisms of action of aminophylline are discussed.Abbreviations cAMP adenosine 3:5-cyclic monophosphate - FC fusicoccin - IAA indole-3-acetic acid - MES 2-N-morpholinoethanesulfonic acid - PDE cyclic nucleotide phosphodiesterase     

Determination of cytoplasmic receptors for specific steroid hormones. II) Androgen receptor

Cytoplasmic receptors for 5 alpha-dihydrotestosterone (3H-DHT) were determined in normal and hypertrophic human prostate using the slightly modified DCC method we previously standardized for 17beta-estradiol-receptor. Incubations were always performed at 0 degree C for 1 hr. Discrimination between 3H-DHT binding to cytoplasmic receptor and to Sex Hormone Binding Globulin (SHBG) was achieved on the basis of binding affinity, thermolability and pattern of specificity by various steroid hormones. In particular, 5 beta-DHT did not bind to cytoplasmic receptor, while it did to SHBG.     

Determination of cytoplasmic receptors for specific steroid hormones. III) Progestin receptor

A method for determining cytoplasmic progesterone receptor was standardized in normal human endometrium comparing two different tracers, 3H-progesterone (3H-P) and 3H-medroxyprogesterone acetate (3H-MAP), a synthetic progestin which does not bind to Corticosteroid Binding Globulin (CBG). Receptor assays were performed as previously reported for 17beta-estradiol receptor, with slight modifications: incubation lasted 1 hr at 0 degree C, followed by 5 min DCC exposure under the same conditions. When 3H-P was employed as tracer, blanks performed with cold MAP gave similar results as using cortisol in incubation tubes and progesterone and cortisol in blanks. 3H-MAP was a good tracer for progesterone receptor because it neither bound to CBG nor to androgen or cortisol receptors; it had very high affinity and specificity for P-R; it was not metabolized by cytosol at 0 degree C and, finally, it detected receptor amounts quite comparable to those obtained using 3H-P.     

Incorporation of 18O2 into brain serotonin in vivo as a procedure for estimating turnover: a feasibility study in animals.

Serotonin (5HT) containing ¹⁸0 instead of ¹⁶0 was found in brain after exposing rats to an atmosphere of ¹⁸0₂. The ¹⁸0 incorporation appeared linear with time and 22% of the 5HT in striatum contained ¹⁸0 after one hour of exposure. ¹⁸0 entered 5HT by enzymatic reaction rather than by isotope exchange as treatment with p-chlorophenylalanine, to block tryptophan hydroxylase, prevented incorporation, while more ¹⁸0-5HT than normal was found in brain after treatment with pargyline, to block monoamine oxidase. Our studies suggest that the incorporation of ¹⁸0₂ into brain 5HT might be a feasible approach for evaluating 5HT turnover in animals and with some modification might be applied in man.     

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.     

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.