Implications of a consensus recognition site for phosphatidylcholine separate from the active site in cobra venom phospholipases A2.

A model structure of Naja naja kaouthia cobra venom phospholipase A2 has been constructed by utilizing molecular modeling techniques. Analysis of the model and available biochemical data reveal the presence in this enzyme of a putative recognition site for choline derivatives in loop 57-70 made up of residues Trp-61, Tyr-63, Phe-64, and Lys-65, together with Glu-55. The magnitude and shape of the electrostatic potential in this binding site are approximately 80% similar to that in the McPC603 antibody binding site specifically recognizing phosphocholine. Docking studies indicate that the recognition site we now describe and the phosphocholine head of an n-alkylphosphocholine molecule are complementary both sterically and electronically, mainly due to anion-cation and cation-pi interactions. Moreover, binding enthalpies of n-heptylphosphocholine to this site are found to parallel the catalytic rate of pancreatic, mutant pancreatic, and cobra venom phospholipase A2 enzymes acting on dihexanoylphosphatidylcholine micelles, suggesting that it behaves as an activator site. This proposal is in keeping with the "dual phospholipid" model put forward to account for the phenomenon of interfacial activation. This novel site is also shown to be able to discriminate choline derivatives from ethanolamine derivatives, in accord with experimental data. On the basis of the results obtained, two functions are assigned to this putative activator site: (i) desolvation of the lipid-enzyme interface, particularly the surroundings of tyrosine at position 69 (Tyr-63), and (ii) opening of the entrance to the active site by means of a conformational change of Tyr-63 whose chi 2 angle rotates nearly 60 degrees.     

Topological mapping of the active sites of cytochromes P4502B1 and P4502B2 by in situ rearrangement of aryl-iron complexes.

Cytochrome P4502B1 reacts with phenylhydrazine or phenyldiazene to give an iron-phenyl complex that oxidatively rearranges in situ to the two N-phenylprotoporphyrin IX regioisomers with the phenyl group on pyrrole rings A (NA) and D (ND) [Swanson, B. A., Dutton, D. R., Lunetta, J. M., Yang, C. S., & Ortiz de Montellano, P. R. (1991) J. Biol. Chem. 266, 19258-19264]. The conclusion that the active site of cytochrome P4502B1 is open above pyrrole rings A and D but not B and C is extended here by studies with larger arylhydrazines. The N-arylprotoporphyrin IX standards required for product identification were obtained by reaction of the arylhydrazines with equine myoglobin. Cytochrome P4502B1 aryl-iron complex formation followed by oxidative shift of the aryl group produces the following N-aryl-protoporphyrin IX NA:ND regioisomer ratios: phenylhydrazine (39:61), 3,5-dimethylphenylhydrazine (29:71), 4-tert-butylhydrazine (25:75), 2-naphthylhydrazine (less than 2:greater than 98), and 4-(phenyl)phenylhydrazine (87:13). Electron-withdrawing substituents (as in 3,5-dichlorophenyl) prevent the aryl group shift. The increase in the proportion of the ND regioisomer with increasing bulk of the aryl group suggests that the region over pyrrole ring A is more sterically encumbered than that over pyrrole ring D. The regiospecificity is reversed, however, with 4-(phenyl)phenylhydrazine, which primarily gives the NA regioisomer. This reversal suggests that the active site has a sloping roof that is higher over pyrrole ring A than pyrrole ring D and that provides a larger steric barrier to the shift of tall aryl moieties than the barrier over pyrrole ring A.(ABSTRACT TRUNCATED AT 250 WORDS)     

Interaction of retinol and retinoic acid with phospholipid membranes. A differential scanning calorimetry study.

The influence of retinol and retinoic acid, two retinoids of major interest, on the main gel to liquid-crystalline phase transition of different phospholipid membranes has been studied by means of differential scanning calorimetry. Both compounds exerted perturbing effects on the phase transition of membranes composed of dipalmitoylphosphatidylcholine or dipalmitoylphosphatidylethanolamine. At concentrations up to 42.5 mol% of retinoid in the membrane, the delta H was not much affected with respect to the pure phospholipid, indicating a rather slight interaction. As the concentration of retinol was increased the Tc transition temperature decreased. A fluid-phase immiscibility was observed for the system DPPC/retinol at concentrations between 0 and 33 mol%. Almost ideal phase diagrams were obtained for the mixture DPPE/retinol. At concentrations of 33 mol% and higher retinol was able to induce phase separations in DPPC membranes, but not in DPPE. The effect of retinoic acid was much weaker, the Tc and delta H remaining almost unaltered and equal to that of the pure phospholipid up to concentrations of 30 mol%, at neutral pH. Retinoic acid exerted a pH-dependent effect. As the pH decreased, and therefore increased the extent of protonation of retinoic acid, the pertubation of the membrane induced by this compound was less. A strong effect, both on Tc and delta H, was observed at pH 10, where the retinoic acid moiety will be mainly unprotonated and the negative charge will generate repulsive forces thus destabilizing the membrane. The mixture DPPC/retinoic acid presents a region of fluid-phase immiscibility. At low pH, when the retinoic acid moiety was fully protonated, this fluid-immiscibility region extended from 0 to 36 mol% of retinoic acid, but its size decreased with increasing pH, and at pH 10 it was only found from 0 to 3 mol%. These results are discussed in terms of the possible retinoid/phospholipid interactions and the disposition of the retinoid moiety in the bilayer.     

Influence of Leaf Age, Soil Moisture, VPD and Time of Day on Leaf Conductance of Various Musa Genotypes in a Humid Forest-Moist Savanna Transition Site

Leaf age effects on the leaf conductance to water vapour diffusionof the adaxial and abaxial leaf surfaces were measured in themorning and in the afternoon on 17 different plantain and banana(Musa spp.) genotypes. The irradiance levels increased three-foldwhile leaf to air vapour pressure deficit levels increased two-to four-fold from morning to afternoon during the sampling periodin a field site located in the humid forest-moist savanna transitionzone of Nigeria. Conductance values were reduced in older, andsenescing leaves relative to the young and mature leaves bothin the morning and in the afternoon. Conductances were higherfor the abaxial leaf surfaces than the adaxial surface and higherin the afternoon than in the morning, with some genotypic differences.Lower values of leaf conductance to water vapour in the afternoonunder a short dry spell was sufficiently variable (P 0·05)among the test genotypes to indicate potential adaptation totransient dry conditions. Differential and relative leaf conductanceadjustments were noted among genotypes experiencing a shortdry spell versus non-limiting soil moisture conditions. Significantgenotypic differences were observed for leaf conductance amongthe 17 genotypes during the afternoon on the lower leaf surfaceof younger leaves. ABB cooking banana cultivars 'Fougamou' and'Bluggoe' might be potentially promising cultivars for transientdry conditions while AAB plantain 'Bobby Tannap' and one ofits hybrids TMPx 582-4 could be very sensitive to short dryspells according to this evaluation.Copyright 1994, 1999 AcademicPress Musa spp., Musa hybrids, adaxial leaf surface, abaxial leaf surface, stomatal response     

Inheritance of black sigatoka disease resistance in plantain-banana (Musa spp.) hybrids

Black sigatoka (Mycosphaerella fijiensis Morelet), an airborne fungal leaf-spot disease, is a major constraint to plantain and banana (Musa spp.) production world-wide. Gaining further knowledge of the genetics of host-plant resistance will enhance the development of resistant cultivars, which is considered to be the most appropriate means to achieve stable production. Genetic analysis was conducted on 101 euploid (2x, 3x and 4x) progenies, obtained from crossing two susceptible triploid plantain cultivars with the resistant wild diploid banana Calcutta 4. Segregating progenies, and a susceptible reference plantain cultivar, were evaluated over 2 consecutive years. Three distinct levels of host response to black sigatoka were defined as follows: susceptible (< 8 leaves without spots), less susceptible (8–10) and partially resistant (> 10). Segregation ratios for resistance at the 2x level fitted a genetic model having one major recessive resistance allele (bs ₁) and two independent alleles with additive effects (bsr ₂ and bsr ₃). A similar model explains the results at the 4x level assuming that the favourable resistance alleles have a dosage effect when four copies of them are present in their respective loci (bs _i ⁴ ). The proposed model was further validated by segregation data of S ₁ progenies. Mechanisms of black sigatoka resistance are discussed in relation to the genetic model.     

Inhibition of the HIV-1 and HIV-2 proteases by curcumin and curcumin boron complexes

Curcumin, a relatively non-toxic natural product isolated from Curcuma longa, is a modest inhibitor of the HIV-1 (1050 = 100 μM) and HIV-2 (IC₅₀ = 250 μM) proteases. Simple modifications of the curcumin structure raise the IC₅₀ value but complexes of the central dihydroxy groups of curcumin with boron lower the IC₅₀ to a value as low as 6 μM. The boron complexes are also time-dependent inactivators of the HIV proteases. The increased affinity of the boron complexes may reflect binding of the orthogonal domains of the inhibitor in intersecting sites within the substrate-binding cavity of the enzyme, while activation of the ,β-unsaturated carbonyl group of curcumin by chelation to boron probably accounts for time-dependent inhibition of the enzyme.     

Mevalonate dependency of the early cell cycle mitogenic response to epidermal growth factor and prostaglandin F2α in Swiss mouse 3T3 cells

Lovastatin (LOV), a hydroxy-methylglutaryl-coenzyme A (HMGCoA) reductase competitive inhibitor, blocks epidermal growth factor (EGF)— or prostaglandin F_2α (PGF_2α)—induced mitogenesis in confluent resting Swiss 3T3 cells. This inhibition occurs even in the presence of insulin, which potentiates the action of these mitogens in such cells. LOV exerts its effect in a 2–80 μM concentration range, with both mitogens attaining 50% inhibition at 7.5 μM. LOV exerted its effect within 0–8 h following mitogenic induction. Mevanolactone (10–80 μM) in the presence of LOV could reverse LOV inhibition within a similar time period. LOV-induced blockage of PGF_2α response is reflected in a decrease in the rate of cell entry into S phase. Neither cholesterol, ubiquinone, nor dolichols of various lengths could revert LOV blockage. In EGF- or PGF_2α-stimulated cells, LOV did not inhibit [³H]leucine or [³H]mannose incorporation into proteins, while tunicamycin, an inhibitor of N′ glycosylation, prevented this last phenomenon. Thus, it appears that LOV exerts its action neither by inhibiting unspecific protein synthesis nor by impairing the N′ glycosylation process. These findings strongly suggest that either EGF or PGF_2α stimulations generate early cell cycle signals which induce mevalonate formation, N′ glycoprotein synthesis, and proliferation. The causal relationship of these events to various mechanisms controlling the onset of DNA synthesis is also discussed. © 1995 Wiley-Liss, Inc.     

Phenotypic Diversity and Patterns of Variation in West and Central African Plantains (Musa Spp., AAB group Musaceae)

Plantains (Musa spp., AAB group) are an important food crop and an integral component of the farming systems in the lowland humid forest zone of West and Central Africa. A group of 24 plantain cultivars, representing the major variability in West Africa, was evaluated for nine quantitative characters. The association between growth and yield parameters in this African plantain germplasm was examined to determine if the pattern of quantitative variation in inflorescence and vegetative traits agreed with taxonomic groupings based on inflorescence type and plant size. Phenotypic correlations between these traits were calculated. Giant cultivars were taller, their pseudostem thicker, and they flowered much later than medium-sized cultivars. Giant cultivars produced more foliage, resulting in heavier bunches with more hands and fruits. Groupings that resulted following principal component analysis (PCA) supported conventional taxonomic groupings of plantains. PC A was based mainly on time to flowering, pseudostem height, and number of fruits. The last two traits, in combination with the number of hermaphrodite flowers and the persistence of the male bud, sufficed to group plantain cultivars.     

Acutely administered melatonin reduces oxidative damage in lung and brain induced by hyperbaric oxygen

Pablos, Marta I., Russel J. Reiter, Jin-Ing Chuang, GenaroG. Ortiz, Juan M. Guerrero, Ewa Sewerynek, Maria T. Agapito, DanielaMelchiorri, Richard Lawrence, and Susan M. Deneke. Acutely administered melatonin reduces oxidative damage in lung and brain induced by hyperbaric oxygen. J. Appl.Physiol. 83(2): 354-358, 1997.Hyperbaric oxygenexposure rapidly induces lipid peroxidation and cellular damage in avariety of organs. In this study, we demonstrate that the exposure ofrats to 4 atmospheres of 100% oxygen for 90 min is associated withincreased levels of lipid peroxidation products [malonaldehyde(MDA) and 4-hydroxyalkenals (4-HDA)] and withchanges in the activities of two antioxidative enzymes[glutathione peroxidase (GPX) and glutathione reductase (GR)], as well as in the glutathione status in the lungs and in the brain. Products of lipid peroxidation increased after hyperbaric hyperoxia, both GPX and GR activities were decreased, and levels oftotal glutathione (reduced+oxidized) and glutathione disulfide (oxidized glutathione) increased in both lung and brain areas (cerebralcortex, hippocampus, hypothalamus, striatum, and cerebellum) but not inliver. When animals were injected with melatonin (10 mg/kg) immediatelybefore the 90-min hyperbaric oxygen exposure, all measurements ofoxidative damage were prevented and were similar to those in untreatedcontrol animals. Melatonin's actions may be related to a variety ofmechanisms, some of which remain to be identified, including itsability to directly scavenge free radicals and its induction ofantioxidative enzymes via specific melatonin receptors.