Factors affecting Citrus tree isozyme-gene expression

Ten enzymatic systems of Citrus species and cultivars have been evaluated for identification purposes and for genetic variability studies. The following factors that could affect their expression were studied: season of sampling, location, rootstock, position of the branch, infection, and age of the tree. Differences involving the presence-absence of the Cu/Zn SOD within the same tree were found. This difference is mainly related to the position of the leaf relative to the sunlight. No change was observed at any of the ten enzymatic systems assayed regarding the location, the rootstock, the growing condition, the season, or the infection with most virus and virus-like pathogens. Viroids induced noticeable changes on 6PG and PRXa zymograms in C. medica. A new peroxidase (not present in healthy plants) was detected that could be related to appearance of symptoms. This may induce errors when trees without sanitary control are characterized by this enzymatic system. On the other hand, it provides a new possibility for studying the plant response to the presence of viroids. An effect of age, from 3 months up to 12 years, was observed on citrange Troyer and mandarin Cleopatra PRX, MDH and 6PG patterns. An important change occurs around the first year, most likely related to the end of the seedling stage. This is followed by a long transition phase, the end of which (around 9 years later) coincides with a change in the PRX pattern. These age-related changes seem to involve post-translational modifications of pre-existing isozymes.     

Chromosome localization and characterization of a family of long interspersed repetitive DNA elements from the genus Zea

This paper describes the characterization and chromosomal distribution of new long repetitive sequences present in all species of the genus Zea. These sequences constitute a family of moderately repetitive elements ranging approximately from 1350 to 1700 copies per haploid genome in modern maize (Zea mays ssp. mays) and teosinte (Zea diploperennis), respectively. The elements are long, probably larger than 9 kb, and they show a highly conserved internal organization among Zea subspecies and species. The elements are present in all maize chromosomes in an interspersed pattern of distribution, are absent from centromeric and pericentric heterochromatin, and with some clustering in the distal regions of chromosome arms.     

Dose-dependent effect of heparin on fertilizing ability of goat spermatozoa

Intact bovine oocytes were used to study the effect of heparin on goat IVF. Oocytes were matured in Medium 199 plus estrous sheep serum. Fresh semen was incubated for 4 h at room temperature, and spermatozoa were then resuspended in medium Talp plus serum and incubated further for 1 h at 39 degrees C in 5% CO(2) in air. Later, spermatozoa were resuspended in Talp plus serum and heparin and were then incubated in microdrops until the oocytes were matured. In Experiment 1, the effect of heparin on spermatozoa from individual males was studied by a dose-response curve. In Experiment 2, the timing of sperm penetration in matured oocytes was studied to assess the stage at which the action of heparin could be expressed in the fertilization process. In Experiment 3, heparin from the same source but at different grades of bioactivity was adjusted for bioactivity and its effect on spermatozoa was compared in terms of penetration rates in order to identify heparin-dependent variations on goat IVF. In Experiment 4, the influence of calcium on the effect of heparin at different levels of bioactivity on the fertilizing ability spermatozoa was assessed as in Experiment 3. In Experiment 5, different batches of heparin from the same source and grade of bioactivity were compared as above. The results suggest that 1) heparin stimulates fertilization rates following a comparable pattern between males; 2) the most probable site of action is at the stage of sperm capacitation; and 3) provided that the source and grade of bioactivity is preserved, heparin maintains the efficiency of sperm penetration into matured oocytes.     

Polysaccharides from the seeds of Senna multijuga

The seeds of Senna multijuga were extracted with water or 1% acetic acid and treated with ethanol, resulting in two insoluble fractions. After purification, the major one (FIA, 23%) was shown to be a galactomannan (Man:Gal 2.3:1;[] = + 54.6;[η]=1340mlg⁻¹). It consists of a main chain of (1 → 4)-linked β-d-mannopyranosyl residues substituted at 06 by single-unit -d-galactopyranosyl side chains. The second fraction (FIB, 2.5%) was an O-acetyl-glucuronoarabinoxylan from the seed coats (O-acetyl 8.3 mol%; glucuronic acid 11.7%, Xyl:Ara ratio 20:1), which showed a predominance of 4-O-substituted Xylp units (84.4%), branched at 03 with non-reducing end units of Xylp, Araf and glucuronic acid. The O-acetyl positions in d-xylosyl units are at 02 (4.8%), 03 (4.4%) and 02,3 (0.9%). The ratio between 03 and 02 determined by ¹³C-nuclear magnetic resonance spectroscopy is 1.5:1.     

The vegetable rennet of Cynara cardunculus L. contains two proteinases with chymosin and pepsin-like specificities

The flowers of cardoon (genus Cynara) are traditionally used in Portugal for cheese making. In this work the vegetable rennet of the species Cynara cardunculus L. was characterized in terms of enzymic composition and proteolytic specificity of its proteinases (cardosin A and cardosin B). Cardosin A was found to cleave insulin B chain at the bonds Leu15-Tyr16, Leu17-Val18 and Phe25-Tyr26. In addition to the bonds mentioned cardosin B cleaves also Glu13-Ala14, Ala14-Leu15 and Phe24-Phe25 indicating that it has a broader specificity. The kinetic parameters for the hydrolysis of the synthetic peptide Leu-Ser-Phe(NO₂)-Nle-Ala-Leu-oMe were also determined and compared to those of chymosin and pepsin. The results obtained indicate that in terms of specificity and kinetic parameters cardosin A is similar to chymosin whereas cardosin B is similar to pepsin. It appears therefore that the enzyme composition of cardoon rennet closely resembles that of calf rennet.     

The binding motifs for Ac transposase are absolutely required for excision of Ds1 in maize

A reverse genetic system for studying excision of the transposable elementDs1 in maize plants has been established previously. In this system, theDs1 element, as part of the genome of maize streak virus (MSV), is introduced into maize plants via agroinfection. In the presence of theAc element, excision ofDs1 from the MSV genome results in the appearance of viral symptoms on the maize plants. Here, we used this system to study DNA sequences requiredin cis for excision ofDs1. TheDs1 element contains theAc transposase binding motif AAACGG in only one of its subterminal regions (defined here as the 5′ subterminal region). We showed that mutation of these motifs abolished completely the excision capacity ofDs1. This is the first direct demonstration that the transposase binding motifs are essential for excision. Mutagenesis with oligonucleotide insertions in the other (3′) subterminal region resulted in elements with either a reduced or an increased excision efficiency, indicating that this subterminal region also has an important function.     

A new mite-plant association: mites living amidst the adhesive traps of a carnivorous plant

Non-antagonistic interactions between arthropods and leaves of insectivorous plants with adhesive traps so far have never been reported. The mites are common prey of such plants, but we have found a new subspecies of the mite Oribatula tibialis living on the leaves of Pinguicula longifolia. Because of its small size and the low glandular density of the host, the mite moves without being trapped by the mucilaginous droplets of the leaf surface. P. longifolia provides shelter and food for the mite, while the plant may also benefit because of its fungivorous and scavenging activities. This new interaction is another dramatic example of widespread miteplant associations.     

Study of interaction between calcium and zinc ond-galactose intestinal transport

Zinc is an essential trace element necessary to life. This metal may exert some of its physiological effects by acting directly on cellular membranes, either by altering permeability or by modulating the activity of membrane-bound enzymes. On the other hand, calcium is an essential element in a wide variety of cellular activities. The aim of the present work was to study a possible interaction between zinc and calcium on intestinal transport ofd-galactose in jejunum of rabbit in vitro. In media with Ca²⁺, when ZnCl₂ was present at 0.5 or 1 mM, zinc was found to reduce thed-galactose absorption significantly. In Ca²⁺-free media, where CaCl₂ was omitted and replaced isotonically with choline chloride, the sugar transport was not modified by zinc. Verapamil at 10⁻⁶ M (blocking mainly Ca²⁺ transport) did not modify the inhibitory effect of zinc ond-galactose transport. When 10⁻⁶ M of A 23187 (Ca²⁺-specific ionophore) was added with/without Ca²⁺ to the media, ZnCl₂ produced no change in sugar transport. These results could suggest a possible interaction of calcium and zinc for the same chemical groups of membrane, which could affect the intestinal absorption of sugars.     

The IleL229 → Met mutation impairs the quinone binding to the QB-pocket in reaction centers of Rhodobacter sphaeroides

A spontaneous mutant (R/89) of photosynthetic purple bacterium Rhodobacter sphaeroides R-26 was selected for resistance to 200 M atrazin. It showed increased resistance to interquinone electron transfer inhibitors of o-phenanthroline (resistance factor, RF=20) in UQ_o reconstituted isolated reaction centers and terbutryne in reaction centers (RF=55) and in chromatophores (RF=85). The amino acid sequence of the Q_B binding protein of the photosynthetic reaction center (the L subunit) was determined by sequencing the corresponding pufL gene and a single mutation was found (Ile^L229 Met). The changed amino acid of the mutant strain is in van der Waals contact with the secondary quinone Q_B. The binding and redox properties of Q_B in the mutant were characterized by kinetic (charge recombination) and multiple turnover (cytochrome oxidation and semiquinone oscillation) assays of the reaction center. The free energy for stabilization of Q_AQ_B ^– with respect to Q_A ^–Q_B was G_AB=–60 meV and 0 meV in reaction centers and G_AB=–85 meV and –46 meV in chromatophores of R-26 and R/89 strains at pH 8, respectively. The dissociation constants of the quinone UQ_o and semiquinone UQ_o ^– in reaction centers from R-26 and R/89 showed significant and different pH dependence. The observed changes in binding and redox properties of quinones are interpreted in terms of differential effects (electrostatics and mesomerism) of mutation on the oxidized and reduced states of Q_B.Abbreviations BChl bacteriochlorophyll - Ile isoleucine - Met methionin - P primary donor - Q_A primary quinone acceptor - Q_B secondary quinone acceptor - RC reaction center protein - UQ_o 2,3-dimethoxy-5-methyl benzoquinone - UQ₁₀ ubiquinone 50 This work is dedicated to the memory of Randall Ross Stein (1954–1994) and is, in a small way, a testament to the impact which Randy's ideas have had on the development of the field of competitive herbicide binding.     

The structural role of the carotenoid in the bacterial light-harvesting protein 2 (LH2) of Rhodonbacter capsulatus. A Fourier transform Raman spectroscopy and circular dichroism study

In previous work (Zurdo J, Fernández-Cabrera C and Ramírez JM (1993) Biochem J 290: 531–537), it had been shown that selective extraction of the carotenoid from the light-harvesting protein 2 (LH2) of Rhodobacter capsulatus induced the dissociation of 800-nm absorbing bacteriochlorophyll (Bchl), a 10-nm red shift of 854-nm Bchl, and a decrease of the stability of the protein in detergent solution. In the present study, the Fourier transform Raman and near-infrared circular dichroism spectra of native and carotenoid-depleted LH2 membrane preparations were compared. It was found that while the coupled carbonyls of 854-nm Bchl remained specifically H-bonded to the peptides after carotenoid extraction, the optical activity of the near-infrared electronic transition was significantly altered. Given the excitonic origin of such optical activity, our data suggest that carotenoid extraction elicits a rearrengement of the chromophore cluster and of the associated polypeptide subunits. This implies a significant role of the carotenoid in maintaining the native quaternary structure of the protein, which would be consistent with the observed dissociation of 800-nm Bchl and the loss of solubilized LH2 stability that result from carotenoid removal. There is no evidence for a similar role of the carotenoid in the LH1 protein.Abbreviations Bchl bacteriochlorophyll - FT Fourier transform - CD circular dichroism - LH1 and LH2 the bacterial light-harvesting proteins 1 and 2 In memoriam of Daniel I. Arnon.