Amino-acids imitate the EDTA activation on the fructose-1,6-bisphosphatase of mantle tissue from the sea mussel (Mytilus galloprovincialis Lmk).

In the absence of AMP and Fru-2,6-P2, several amino-acids such as histidine, lysine, alanine, aspartic acid, and other molecules, as reduced glutathione or citrate, activate FBPase-1 from Mytilus galloprovincialis mantle. AMP decreases Vmax and Km for Fru-1,6-P2 both in the absence and in the presence of activators; but the addition of Fru-2,6-P2 decreases the affinity of the enzyme by its substrate. Na+ acts as a inhibitor reducing both Vmax and Km. The Km value is lower than the physiological level of Fru-1,6-P2, suggesting that the enzyme is operative but its activity is very reduced.     

Direct gene transfer into Actinidia deliciosa protoplasts: analysis of transient expression of the CAT gene using TLC autoradiography and a GC-MS-based method

Chloramphenicol acetyl transferase (CAT) gene was used as a reporter gene to assess the conditions for polyethylene glycol (PEG)-mediated transfection of kiwifruit protoplasts. The effect of plasmid concentration and the presence of carrier DNA were each assessed by analysing CAT activity in transfected protoplasts using thin-layer chromatography (TLC) autoradiographic detection of acetylated chloramphenicol. A gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) non-radioactive method was developed for monitoring CAT gene activity. This method provides a high speed of analysis (30 min) and precise means of detecting acetylated products at the nanomolar level, enabling quantification at very low transfection rates. Using this method we optimized plasmid and PEG concentration and also assessed the effect of heat shock on transfection. The best CAT activity was obtained using 30% polyethylene glycol 4000 and by submitting protoplasts to heat shock (45 °C, 5 min) prior to transfection.     

Morphogenesis in leaf,hypocotyl and root explants of Digitalis thapsi L. cultured in vitro

The effects of the auxins 2,4-D, NAA and IAA either alone or in combination with kinetin or BA were investigated to assess the morphogenetic potential of leaf, root and hypocotyl explants of Digitalis thapsi. Calluses were obtained from the three explants in basal medium without the addition of growth regulators and in leaves, the calluses formed roots. Application of 2,4-D, NAA or BA increased callus formation. The presence of NAA induced root formation and that of BA induced shoot formation via callus interphase. Indole-3-acetic acid alone only induced the generation of roots in the hypocotyl callus. Kinetin was ineffective in all the explants tested. Combinations of NAA with kinetin or BA were more effective in inducing organogenesis in leaf explants. Optimum responses were obtained in hypocotyl and root explants by using IAA in combination with BA, the highest rate of shoot regeneration being observed in hypocotyl explants.Rooting of the differentiated shoots was readily achieved in media without growth regulators. Regenerated plantlets were transferred to soil and grew with a survival rate of 70%.Abbreviations BA benzyladenine - 2,4-D 2,4-dichlorophenoxyacetic acid - IAA indoleacetic acid, Kin-kinetin - NAA naphthaleneacetic acid     

The effect of indole-3-acetylaspartic acid on adventitious root formation on bean cuttings

The influence of indole-3-acetylaspartic acid (IAAsp) on rooting of stem cuttings from bean plants (Phaseolus vulgaris L.) of different ages, cultivated at different temperatures (17°, 21° and 25°C) was studied and compared to that of indole-3-acetic acid (IAA). At a concentration of 10^–4 M, IAAsp only nonsignificantly stimulated adventitious root formation, approximately to the same level as IAA in all treatments. IAAsp at 5×10^–4 M further enhanced rooting, by up 200% of control values, with little influence of temperature conditions and stock plant age. This concentration of IAA usually stimulated rooting more than the conjugate. The largest differences between the effects of IAAsp and IAA occured at the highest cultivation temperature of 25°C where stock plant age also influenced the response. The number of roots produced in comparison with the control, was enhanced from 350% on cuttings from the youngest plants to more than 600% on cuttings from the oldest. In contrast to the conjugate, 5×10^–4 M IAA induced hypocotyl swelling and injury of the epidermis at the base of cuttings, in all treatments.     

Computational study of radiation chemical processing in comet nuclei

Cometary nuclei have been exposed to high levels of ionizing radiation since their formation. We present here some results of a computer model calculation of the effect of ionizing radiation on cometary material. The external (cosmic rays) and internal (embedded radionuclides) contributions in the processing of cometary nuclei are considered. As a first approximation we have used the available kinetic data of the liquid water system to model the radiation effects in a frozen cometary environment. Out data suggest that massive radiation chemical processing due to cosmic rays may have taken place only in the outer layers of comets. The internal contribution of radionuclides to the radiation processing of comet cores seems to be modest. Therefore, comets could be carriers of intact homochiral biomolecules.Part of this work was carried out during a leave at the Laboratory of Chemical Evolution.     

Experimental and computational study of the radiation-induced decomposition of formaldehyde. Implications to cometary nuclei

The radiation-induced decomposition of aqueous solutions of formaldehyde was studied at 298 and 77 K in order to obtain an insight into the possible role of ionizing radiation on cometary chemistry. Aqueous solutions of 1.0 mol dm^–3 formaldehyde were exposed to -radiation in the dose range from 0.01 kGy to 1.2 MGy. The radiation chemical yield of decomposition of formaldehyde was determined to be: G(–CH₂(OH)₂)=26.3±1.2 at 298 K and G0.48 at 77 K.Based on previous estimates of the total dose of ionizing radiation that comets have accumulated over 4.6 billion years, we predict a radiation-induced depletion of formaldehyde as a function of depth in comet nuclei: 100% destruction in the outer layers (0–20 m) and 10% destruction in the interior layers.A Portion of this work was presented at the XXVIII COSPAR Plenary Meeting (MF. 7) held at the Hague, The Netherlands on June 25–26, 1990.     

A guinea-pig hereditary cataract contains a splice-site deletion in a crystallin gene

A congenital cataract present in guinea pigs provided a unique opportunity to study a hereditary lens diseases at the molecular level. ζ-crystallin, one of the most abundant guinea pig lens proteins, was found to be altered in the lens of cataractous animals. Several ζ-crystallin cDNA clones were isolated from a cataractous lens library and found to contain a 102-bp deletion towards the 3′ end of the coding region. The deletion does not interfere with the reading frame but results in a protein 34 amino acids shorter. Sequence analysis of a normal genomic ζ-crystallin clone revealed that the missing 102-bp fragment corresponds to an entire exon (exon 7). PCR analysis of the genomic DNA isolated from cataractous animals showed that exon 7, though missing from the mRNA, is intact in the cataractous genome. Further sequence analysis of the α-crystallin gene disclosed a dinucleotide delection of the universal AG at the acceptor splice-site of intron 6 of the mutant gene. The presence of this mutation results in the skipping of exon 7 during the mRNA processing which in turn results in the altered ζ-crystallin protein. This if the first time a genomic mutation in an enzyme/crytallin gene has been directly linked to a congenital cataract.     

Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions

Summary A novel approach to tailored selective excitation for the measurement of NMR spectra in non-deuterated aqueous solutions (WATERGATE, WATER suppression by GraAdient-Tailored Excitation) is described. The gradient echo sequence, which effectively combines one selective 180° radiofrequency pulse and two field gradient pulses, achieves highly selective and effective water suppression. This technique is ideally suited for the rapid collection of multi-dimensional data since a single-scan acquisition produces a pure phase NMR spectrum with a perfectly flat baseline, at the highest possible sensitivity. Application to the fast measurement of 2D NOE data of a 2.2. mM solution of a double-stranded DNA fragment in 90% H₂O at 5 °C is presented.