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.     

Factors controlling somatic embryogenesis

Histological and ultrastructural, molecular and elemental distribution changes were investigated during the induction of direct somatic embryogenesis using theCamellia japonica leaf culture system. In this culture system, direct somatic embryogenesis is induced in a controlled way in a specific leaf region (leaf blade) within a leaf. Embryogenic and non-embryogenic leaf regions have characteristic energy-dispersive X-ray spectra already before induction. According to these results electron probe X-ray microanalysis (EPMA) can be a tool for early diagnosis of embryogenic competence. Histological studies showed that severe fluctuations in the number of calcium oxalate crystals and in starch accumulation occur after induction but only in induced tissues. Changes in the cell wall composition of competent cells occur shortly after the induction treatment. The induction of morphogenesis is linked to the appearance of callose covering the surface cells of induced leaves and calluses. A 2^nd deposition of material (cutin) is necessary for normal somatic embryogenesis to occur. The involvement of lipid transfer proteins in the appearance of cutin in the embryogenic regions of the explant is suggested.     

Influence of external factors on secondary embryogenesis and germination in somatic embryos from leaves of Quercus suber

Somatic embryogenesis was obtained in cultures of leaves from young seedlings of Quercus suber L. A two-stage process, in which benzyladenine and naphthaleneacetic acid were added first at high and then at low concentrations, was required to initiate the process. Somatic embryos arose when the explants were subsequently placed on medium lacking plant growth regulators. The embryogenic lines remained productive, by means of secondary embryogenesis, on medium without growth regulators. However, this repetitive induction was influenced by the macronutrient composition of the culture medium. Both low total nitrogen content and high reduced nitrogen concentration decreased the percentage of somatic embryos that showed secondary embryogenesis. Our results suggest that alternate culture on medium that increases embryo proliferation and a low salt medium prohibiting embryo formation will partially synchronize embryo development. Chilling slightly reduced secondary embryogenesis but gave a modest increase in germination. Maturation under light followed by storage at 4 °C for at least 30 days gave the best results in switching embryos from an embryogenic pathway to a germinative one. Under these conditions 15% of embryos showed coordinated root and shoot growth and 35% formed either shoots or mostly roots. These percentages were higher than those of embryos matured in darkness. This result indicates that a specific treatment is required after maturation and before chilling to activate the switch from secondary embryo formation to germination.Abbreviations BA benzyladenine - NAA naphthaleneacetic acid - 2,4-D 2,4-dichlorophenoxyacetic acid - BA indolebutyric acid - MS Murashige & Skoog (1962) medium - SH Schenk & Hildebrandt (1972) medium - G Gamborg (1966, PRL-4-C) medium (macronutrients in mg l^–1: NaH₂PO₄·H₂O, 90; Na₂HPO₄, 30; KCl, 300; (NH₄)₂SO₄, 200; MgSO₄·7H₂O, 250; KNO₃, 1000, CaCl₂·2H₂O, 150) - PGR plant growth regulator     

Analysis of vertical ozone and nitrogen oxides profiles in aPrunus cerasifera canopy

An automatic system was installed for continuous analyses of ozone, sulphur dioxide, nitrogen monoxide and nitrogen dioxide in an experimental orchard with a canopy ofPrunus cerasifera plants in summer 1993. Air samples from three elevations (0.8 m, 1.6 m and 3 m above ground) were sequentially analyzed. Ozone concentrations above the canopy were usually higher than within the canopy; their relationships with stomatal resistance have been investigated. Sulphur dioxide levels were negligible. Nitrogen oxides showed a complex profile, with no particular trend, likely due to a reciprocal exchange between the atmosphere and the ground surface.     

Synthesis and biological evaluation of novel NK-1 tachykinin receptor antagonists: The use of cycloalkyl amino acids as a template

In the course of a program aimed at synthesizing novel, potent NK-1 tachykinin receptor antagonists, we developed upon a bioactive model by comparing the low energy structures of a series of peptide and nonpeptide Substance P antagonists. The comparison was based on the super imposition of the aromatic rings, assuming that the rest of the molecule behaves predominantly as a template to arrange the key aromatic groups in the right spatial position. A series of 2-aminocyclohexane carboxylic acid analogues were then selected as the best templates for reproducing the postulated bioactive structure, leading to several pseudo-peptides with interesting biological activity. According to the molecular modeling, these compounds exhibit a neat parallel facing of the indolyl and naphthyl groups at about 3 Å distance. Ultraviolet absorption and steady state fluorescence measurements support this conclusion, showing a linear correlation between the spectral properties and the binding affinity of these analogues. Stacking of the indole ring with naphthalene gives rise to a complex characterized by a well-defined molar extinction coefficient. Consistently, steady state and lifetime fluorescence measurements suggest that the quenching process is ascribable to ground-state interactions between the chromophores. Implications of the π stacking propensity of aromatic groups in the biological activity of the compounds examined are briefly discussed. © 1995 John Wiley & Sons, Inc.     

Simultaneous determination of NO and NO in fisheries wastewaters high in chloride concentration by capillary ion electrophoresis with direct UV detection

The simultaneous determination by capillary ion electrophoresis of nitrate and nitrite in wastewaters with chloride concentrations of 15 to 23g/l is described. Chloride concentrations over 200mg/l hampered the determination; thus, lead, mercuric and silver acetate were used to precipitate chloride. Silver acetate added in 1.5 times the stoichiometric amount for AgCl formation gave the best results in terms of nitrate and nitrite peak resolution.     

Maintenance of transposable element copy number in natural populations of Drosophila melanogaster and D. simulans

To investigate the main forces controlling the containment of transposable elements (TE) in natural populations, we analyzed the copia, mdg1, and 412 elements in various populations of Drosophila melanogaster and D. simulans. A lower proportion of insertion sites on the X chromosome in comparison with the autosomes suggests that selection against the detrimental effects of TE insertions is the major force containing TE copies in populations of Drosophila. This selection effect hypothesis is strengthened by the absence of the negative correlation between recombination rate and TE copy number along the chromosomes, which was expected under the alternative ectopic exchange model (selection against the deleterious rearrangements promoted by recombination between TE insertions). A cline in 412 copy number in relation to latitude was observed among the natural populations of D. simulans, with very high numbers existing in some local populations (around 60 copies in a sample from Canberra, Australia). An apparent absence of selection effects in this Canberra sample and a value of transposition rate equal to 1–2 × 10^-3 whatever the population and its copy number agree with the idea of recent but temporarily drastic TE movements in local populations. The high values of transposition rate in D. simulans clearly disfavor the hypothesis that the low amount of transposable elements in this species could result from a low transposition rate. This revised version was published online in August 2006 with corrections to the Cover Date.     

Effect of lipids on activity and conformation of lysolecithin:lysolecithin acyltransferase from rabbit lung

Summary Lysolecithin:lysolecithin acyltranferase is an enzyme which in several previous studies has shown a dual behavior catalyzing two types of reaction, transacylation or hydrolysis, with the same substrate. Both activities have shown to be dependent on several environmental conditions and among them, the presence of lipids.The addition of several classes of lipids activated in all the cases the enzyme, decreasing the hydrolysis/transacylation molar ratio. This effect was higher for PC/PE/Chol mixture than for other lipids assayed. Circular dichroism spectra of the enzyme did not show any change with the addition of lipids, concluding that the effect of lipids was not due to any structural change in the protein. The hypothesis has been made of an influence of lipids on the physical state of the substrate as well as, possibly, on the enzyme-substrate interaction.The significance of these effects on the physiological role of lysolecithin:lysolecithin acyltransferase from soluble fraction of rabbit lung is discussed.Abbreviations Chol cholesterol - CMC critical micellar concentration - DPPC dipalmitoylphosphatidylcholine - FA fatty acid - H/T hydrolysis/transacylation molar ratio - LPC lysophosphatidylcholine - PC phosphatidylcholine - PE phosphatidylethanolamine - TG triglyceride - UV ultraviolet