Growth and shoot formation in protoplast-derived calli of Brassica oleracea ssp. acephala and ssp. capitata

The effect of media components and environmental factors on growth and organogenesis of protoplast-derived calli of curly kale and cabbage were tested. Optimal growth (fresh weight increase of calli, shoots and roots) was found at 60 mM sucrose. Lower sucrose concentrations (3–30 mM) were favourable for shoot formation. Nitrate concentrations from 23 to 100 mM in combination with 8 or 21 mM ammonium were optimal for shoot formation. However, growth was reduced by high (100 mM) nitrate concentration. The effects of various organic nitrogen compounds at 0.5 and 2 mM were tested. Glutamine did not influence shoot formation and barely growth. Proline at 0.5 mM stimulated growth of cabbage calli but decreased growth of curly kale calli, and at 2 mM, proline also inhibited shoot production. Adenine sulphate decreased growth of cabbage calli at 0.5 mM, and at 2 mM shoot production was also reduced. Spermidine and spermine inhibited both growth and differentiation. Putrescine resulted in about 50% higher fresh weights, and also increased the number of calli producing shoots by about 35%. More calli produced shoots in white light than in blue or red light or in darkness. The length of the photoperiod or intensity of light was not critical for shoot production.     

Comparative studies of diurnal variations of nitrate reductase activity in wheat, oat and barley

Diurnal variations of in vitro and in vivo (intact tissue assay) nitrate reductase (EC 1.6.6.1) activity and stability were examined in leaves of wheat ( Triticum aestivum L. cv. Runar), oat ( Avcna saliva L. cv. Mustang) and barley ( Hordeum vulgure L. cv. Agneta and cv. Gunillu). Nitrate reductase activity was generally higher for wheat than for oat and barley. However, the diurnal variations of nitrate reductase activity and stability were principally the same for all species, e.g. the high activity during the photoperiod was associated with low stability. All species showed a rapid (30-60 min) increase in the in vitro and in vivo activity when the light was switched on. When light was switched off the in vitro activity decreased rapidly whereas decrease in in vivo activity was slower. These experiments support the hypothesis that an activation/ deactivation mechanism is involved in the regulation of diurnal variations in nitrate reductase activity. Red light enhanced nitrate reductase activity in etiolated wheat and barley leaves. In green leaves, however, the daily increase in nitrate reductase activity was not induced by a brief red light treatment. Indications of different regulation mechanisms for the diurnal variations of nitrate reductase activity among the cereals were not found.     

Conformation of human fibrinogen in solution from polarized triplet spectroscopy.

The rotational motions of human fibrinogen in solution at 20 degrees C have been examined, in the 0.2-12-microseconds time range, by measuring the laser-induced dichroism of the triplet state of an erythrosin probe covalently bonded to the protein. The decay of the anisotropy was multiexponential, and up to three correlation times (phi 1 = 380 +/- 50 ns, phi 2 = 1.1 +/- 0.1 microseconds, and phi 3 = 3.3 +/- 0.6 microseconds) were needed to obtain a satisfactory analysis. The experimental data are consistent with the brownian motions of an elongated, rigid particle. If the correlation times are combined with previous data on the intrinsic viscosity of fibrinogen, the rotational and translational diffusive properties of the protein can be reproduced with high accuracy by idealizing it as an elongated ellipsoid of revolution with dimensions (2a x 2b) of (54 +/- 6) x (7.2 +/- 0.5) nm, having rotational diffusion constants of D parallel = (6.2 +/- 0.7) x 10(5) s-1 and D perpendicular = (5 +/- 1) x 10(4) s-1. The possibility of Ca(2+)-dependent changes in the rigidity or conformation of fibrinogen was excluded by examining the submicrosecond time-resolved fluorescence depolarization of 1-methylpyrene conjugates of the protein in the presence of different calcium concentrations. Although there are inherent difficulties to extrapolate the data on isolated fibrinogen molecules to the polymerizing species, this relatively stiff conformation meets the requirements of the classical half-staggered double-stranded model of fibrin polymerization rather better than those of the recently proposed interlocked single-stranded mechanism.     

Supramolecular zippers elicit interbilayer adhesion of membranes producing cell death

Background

The fluorescent dye 10-N-nonyl acridine orange (NAO) is widely used as a mitochondrial marker. NAO was reported to have cytotoxic effects in cultured eukaryotic cells when incubated at high concentrations. Although the biochemical response of NAO-induced toxicity has been well identified, the underlying molecular mechanism has not yet been explored in detail.

Characterization of Nitrate Reductase from Light- and Dark-Exposed Leaves (Comparison of Different Species and Effects of 14-3-3 Inhibitor Proteins)

Nitrate reductase (NR) was extracted and partially purified from leaves of squash (Curcurbita maxima), spinach (Spinacia oleracea), and three transgenic Nicotiana plumbaginifolia leaves in the presence of phosphatase inhibitors to preserve its phosphorylation state. Purified squash NR showed activation by substrates (hysteresis) when prepared from leaves in the light as well as in darkness. A 14-3-3 protein known to inhibit phosphorylated spinach NR in the presence of Mg2+ decreased by 70 to 85% the activity of purified NR from dark-exposed leaves, whereas NR from light-exposed leaves decreased by 10 to 25%. Apparent lack of posttranslational NR regulation in a transgenic N. plumbaginifolia expressing an NR construct with an N-terminal deletion ([delta]NR) may be explained by more easy dissociation of 14-3-3 proteins from [delta]NR. Partially purified [delta]NR was, however, inhibited by 14-3-3 protein, and the binding constant of 14-3-3 protein (4 x 108 M-1) and the NR-inhibiting protein concentration that results in a 50% reduction of free NR (2.5 nM) were the same for NR and [delta]NR. Regulation of NR activity by phosphorylation and binding of 14-3-3 protein was a general feature for all plants tested, whereas activation by substrates as a possible regulation mechanism was verified only for squash.     

Protein structure probed by polarization spectroscopy. I. Evidence for fibrinogen rigidity from stationary fluorescence

Two fluorescent derivatives of human fibrinogen have been synthesized, by the covalent bonding of 1-dimethylaminoaphthalene-5-sulphonyl and methylpyrene chromophores, to investigate the internal molecular dynamics of the protein in solution. The stationary fluorescence depolarization of these derivatives under isothermal conditions is described here while in an accompanying paper (part II) a time-resolved study is reported. From the static fluorescence data it is concluded that reorientational processes in the subnanosecond and microsecond time ranges account for all the observed depolarization. The faster motion was assigned to the restricted, localized oscillations of the label while the slow motion was ascribed to the overall rotation of the protein molecule. Consequently, the protein in solution appears considerably rigid in the 10-1000 ns range, in contrast with a previous conception of a flexible fibrinogen based on non-isothermal depolarization experiments. These previous experiments are, in fact, concordant with the rigid fibrinogen proposed here if they are reinterpreted using Weber's early ideas on thermally activated depolarization (G. Weber, J. Biochem. 51 (1952) 145).     

Evaluation of Metarhizium anisopliae (Metsch) Sorok. to target larvae and adults of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) in soil and fiber band applications

The aim of this work has been to evaluate in the laboratory the potential of entomopathogenic fungi against adults and larvae of Capnodis tenebrionis (L.) (Coleoptera: Buprestidae) through fiber band application and a potted plant bioassay with soil application, respectively. Our previous findings revealed that Metarhizium anisopliae EAMa 01/58-Su isolate was the most virulent against neonate larvae of the buprestid. In the present work, M. anisopliae EAMa 01/58-Su isolate has been also shown to be highly virulent against adult beetles by immersion in a conidial suspension; thus it was selected to accomplish our objectives. When adult beetles were stimulated to climb 100 x 200 mm non-woven commercial fiber bands impregnated with conidia of M. anisopliae EAMa 01/58-Su isolate, total mortality rates varied from 85.7% to 100.0%; whereas no significant correlation was detected between the time needed to cross the band (mean value 648.7+/-22.4s) and the time of death, with mean average survival time ranging between 10.3 and 16.0 days, compared to 28 days of the controls. Potted seedlings (5-6 months old) of cherry plum (Prunus myrobalana Lois.), a commonly used apricot rootstock, were used to study the efficacy of soil treatment with M. anisopliae EAMa 01/58-Su isolate against neonate C. tenebrionis larvae. The soil inoculation with M. anisopliae EAMa 01/58-Su isolate had a significant effect on the mean number of dead larvae recovered from the roots, with mean mortality ranging from 83.3% to 91.6%; whereas no significant differences were detected between the three fungal doses. In all cases, dead larvae found within roots exhibited external signs of fungal growth. Hence, it may be possible to use M. anisopliae EAMa 01/58-Su isolate in a biocontrol strategy targeting both adults and larvae of C. tenebrionis.