Thr-E11 regulates O2 affinity in Cerebratulus lacteus mini-hemoglobin

The mini-hemoglobin from Cerebratulus lacteus (CerHb) belongs to a class of globins containing the polar Tyr-B10/Gln-E7 amino acid pair that normally causes low rates of O2 dissociation and ultra-high O2 affinity, which suggest O2 sensing or NO scavenging functions. CerHb, however, has high rates of O2 dissociation (kO2 = 200-600 s(-1)) and moderate O2 affinity (KO2) approximately 1 microm(-1)) as a result of a third polar amino acid in its active site, Thr-E11. When Thr-E11 is replaced by Val, kO2 decreases 1000-fold and KO2 increases 130-fold at pH 7.0, 20 degrees C. The mutation also shifts the stretching frequencies of both heme-bound and photodissociated CO, indicating marked changes of the electrostatic field at the active site. The crystal structure of Thr-E11 --> Val CerHbO2 at 1.70 A resolution is almost identical to that of the wild-type protein (root mean square deviation of 0.12 A). The dramatic functional and spectral effects of the Thr-E11 --> Val mutation are due exclusively to changes in the hydrogen bonding network in the active site. Replacing Thr-E11 with Val "frees" the Tyr-B10 hydroxyl group to rotate toward and donate a strong hydrogen bond to the heme-bound ligand, causing a selective increase in O2 affinity, a decrease of the rate coefficient for O2 dissociation, a 40 cm(-1) decrease in nuCO of heme-bound CO, and an increase in ligand migration toward more remote intermediate sites.     

Crystal structure of pseudomonas aeruginosa lipase in the open conformation. The prototype for family I.1 of bacterial lipases

The x-ray structure of the lipase from Pseudomonas aeruginosa PAO1 has been determined at 2.54 A resolution. It is the first structure of a member of homology family I.1 of bacterial lipases. The structure shows a variant of the alpha/beta hydrolase fold, with Ser(82), Asp(229), and His(251) as the catalytic triad residues. Compared with the "canonical" alpha/beta hydrolase fold, the first two beta-strands and one alpha-helix (alphaE) are not present. The absence of helix alphaE allows the formation of a stabilizing intramolecular disulfide bridge. The loop containing His(251) is stabilized by an octahedrally coordinated calcium ion. On top of the active site a lid subdomain is in an open conformation, making the catalytic cleft accessible from the solvent region. A triacylglycerol analogue is covalently bound to Ser(82) in the active site, demonstrating the position of the oxyanion hole and of the three pockets that accommodate the sn-1, sn-2, and sn-3 fatty acid chains. The inhibited enzyme can be thought to mimic the structure of the tetrahedral intermediate that occurs during the acylation step of the reaction. Analysis of the binding mode of the inhibitor suggests that the size of the acyl pocket and the size and interactions of the sn-2 binding pocket are the predominant determinants of the regio- and enantio-preference of the enzyme.     

Screening of some Romanian plants for their activity against medically important insects

The effects of the action of extracts from 82 plant species included in 39 families of indigenous flora against the larvae and adults of house fly (Musca domestica), the mosquitoes (Aedes aegypti and Anopheles atroparvus) and the German cockroach (Blattella germanica) are presented. Some of the extracts were prepared from air-dried and ground plant material which was exhaustively extracted with successive solvents of different polarity: ethyl ether, ethyl alcohol and water. Other extracts were prepared either by distillation of the whole water extracts of 24 hours macerated plants or by water extraction from residues of plants remained after distillation. These plant extracts act as toxicants, growth and development and reproduction inhibitors and repellents. The differential responses induced by these plant extracts on assessed insects were influenced by several factors such as the plant species, the solvents used for extractions, the species and the stages of insect life and also the methods employed for evaluation. Some of the tested plants appear to have a great potential for providing safer insect control agents.     

Water stress-induced modifications of leaf hydraulic architecture in sunflower: co-ordination with gas exchange

The hydraulic architecture, water relationships, and gas exchange of leaves of sunflower plants, grown under different levels of water stress, were measured. Plants were either irrigated with tap water (controls) or with PEG600 solutions with osmotic potential of -0.4 and -0.8 MPa (PEG04 and PEG08 plants, respectively). Mature leaves were measured for hydraulic resistance (R(leaf)) before and after making several cuts across minor veins, thus getting the hydraulic resistance of the venation system (R(venation)). R(leaf) was nearly the same in controls and PEG04 plants but it was reduced by about 30% in PEG08 plants. On the contrary, R(venation) was lowest in controls and increased in PEG04 and PEG08 plants as a likely result of reduction in the diameter of the veins' conduits. As a consequence, the contribution of R(venation) to the overall R(leaf) markedly increased from controls to PEG08 plants. Leaf conductance to water vapour (g(L)) was highest in controls and significantly lower in PEG04 and PEG08 plants. Moreover, g(L) was correlated to R(venation) and to leaf water potential (psi(leaf)) with highly significant linear relationships. It is concluded that water stress has an important effect on the hydraulic construction of leaves. This, in turn, might prove to be a crucial factor in plant-water relationships and gas exchange under water stress conditions.     

Xylem cavitation in the leaf of Prunus laurocerasus and its impact on leaf hydraulics

This paper reports how water stress correlates with changes in hydraulic conductivity of stems, leaf midrib, and whole leaves of Prunus laurocerasus. Water stress caused cavitation-induced dysfunction in vessels of P. laurocerasus. Cavitation was detected acoustically by counts of ultrasonic acoustic emissions and by the loss of hydraulic conductivity measured by a vacuum chamber method. Stems and midribs were approximately equally vulnerable to cavitations. Although midribs suffered a 70% loss of hydraulic conductance at leaf water potentials of -1.5 MPa, there was less than a 10% loss of hydraulic conductance in whole leaves. Cutting and sealing the midrib 20 mm from the leaf base caused only a 30% loss of conduction of the whole leaf. A high-pressure flow meter was used to measure conductance of whole leaves and as the leaf was progressively cut back from tip to base. These data were fitted to a model of hydraulic conductance of leaves that explained the above results, i.e. redundancy in hydraulic pathways whereby water can flow around embolized regions in the leaf, makes whole leaves relatively insensitive to significant changes in conductance of the midrib. The onset of cavitation events in P. laurocerasus leaves correlated with the onset of stomatal closure as found recently in studies of other species in our laboratory.     

Hydrogen peroxide as a mediator of programmed cell death in the blastocyst

Previous work identified in blastocele fluid a soluble activity which killed embryonal carcinoma cells with trophectodermal potential but not those with embryonic potential [35]. From use of a malignant caricature of the late blastocyst, this toxic activity was postulated to be H2O2 [8]. The purpose of this paper was to determine if blastocele fluid also contained amounts of H2O2 capable of mediating the preferential killing of malignant pretrophectodermal cells (ECa 247). We not only observed that blastocele fluid is not toxic for these cells in the presence of catalase, but that malignant cells with embryonic potential (P19) that normally survive exposure to blastocele fluid become sensitive to it if their intracellular glutathione levels are lowered. Thus, it is concluded that the blastocyst contains amounts of H2O2 toxic to malignant pretrophectodermal cells and that glutathione-dependent mechanisms protect malignant inner cell mass cells with embryonic potential. Apparently, H2O2 production and glutathione-dependent protection mechanisms are developmentally regulated in the inner cell mass. These results are discussed with regards to apoptosis and the regulation of tissue mass.     

Structural Bases for the Regulation of CO Binding in the Archaeal Protoglobin from Methanosarcina acetivorans

Studies of CO ligand binding revealed that two protein states with different ligand affinities exist in the protoglobin from Methanosarcina acetivorans (in MaPgb*, residue Cys(E20)101 was mutated to Ser). The switch between the two states occurs upon the ligation of MaPgb*. In this work, site-directed mutagenesis was used to explore the role of selected amino acids in ligand sensing and stabilization and in affecting the equilibrium between the “more reactive” and “less reactive” conformational states of MaPgb*. A combination of experimental data obtained from electronic and resonance Raman absorption spectra, CO ligand-binding kinetics, and X-ray crystallography was employed. Three amino acids were assigned a critical role: Trp(60)B9, Tyr(61)B10, and Phe(93)E11. Trp(60)B9 and Tyr(61)B10 are involved in ligand stabilization in the distal heme pocket; the strength of their interaction was reflected by the spectra of the CO-ligated MaPgb* and by the CO dissociation rate constants. In contrast, Phe(93)E11 is a key player in sensing the heme-bound ligand and promotes the rotation of the Trp(60)B9 side chain, thus favoring ligand stabilization. Although the structural bases of the fast CO binding rate constant of MaPgb* are still unclear, Trp(60)B9, Tyr(61)B10, and Phe(93)E11 play a role in regulating heme/ligand affinity.     

Non-covalent and covalent modifications modulate the reactivity of monomeric mammalian globins

Multimeric globins (e.g., hemoglobin) are considered to be the prototypes of allosteric enzymes, whereas monomeric globins (e.g., myoglobin; Mb) usually are assumed to be non-allosteric. However, the modulation of the functional properties of monomeric globins by non-covalent (or allosteric) and covalent modifications casts doubts on this general assumption. Here, we report examples referable to these two extreme mechanisms modulating the reactivity of three mammalian monomeric globins. Sperm whale Mb, which acts as a reserve supply of O₂ and facilitates the O₂ flux within a myocyte, displays the allosteric modulation of the O₂ affinity on lactate, an obligatory product of glycolysis under anaerobic conditions, thus facilitating O₂ diffusion to the mitochondria in supporting oxidative phosphorylation. Human neuroglobin (NGB), which appears to protect neurons from hypoxia in vitro and in vivo, undergoes hypoxia-dependent phosphorylation (i.e., covalent modulation) affecting the coordination equilibrium of the heme-Fe atom and, in turn, the heme-protein reactivity. This facilitates heme-Fe-ligand binding and enhances the rate of anaerobic nitrite reduction to form NO, thus contributing to cellular adaptation to hypoxia. The reactivity of human cytoglobin (CYGB), which has been postulated to protect cells against oxidative stress, depends on both non-covalent and covalent mechanisms. In fact, the heme reactivity of CYGB depends on the lipid, such as oleate, binding which stabilizes the penta-coordination geometry of the heme-Fe atom. Lastly, the reactivity of NGB and CYGB is modulated by the redox state of the intramolecular CysCD7/CysD5 and CysB2/CysE9 residue pairs, respectively, affecting the heme-Fe atom coordination state. In conclusion, the modulation of monomeric globins reactivity by non-covalent and covalent modifications appears a very widespread phenomenon, opening new perspectives in cell survival and protection. This article is part of a Special Issue entitled: Oxygen Binding and Sensing Proteins.     

Water relations of six sclerophylls growing near Trieste (Northeastern Italy): has sclerophylly a univocal functional significance?

Abstract

The annual time course of the water relations of six sclerophylls has been studied with the aim of: a) defining strategies adopted for withstanding summer water stress and b) to check whether their common sclerophyllous habitus could represent a case of convergent evolution devoted to a univocal functional role. In particular, Phillyrea latifolia L. showed to behave like a drought tolerant as indicated by deep summer drop in leaf water potential (Ψ₁) to near the full turgor loss point (Ψ_lp) and in leaf relative water content (RWC) as caused by xylem cavitation. Prunus laurocerasus L. and Laurus nobilis L. showed to be drought avoiding water spenders in that their Ψ₁ dropped in summer causing prompt recovery in RWC. This was made possible by low cavitation in their twigs. Ilex aquifolium L. was a typical drought avoiding water saver in that both Ψ₁ and RWC remained at very high levels throughout the year. Quercus ilex L. behaved like a drought avoiding water spender which switched to drought tolerance in the warmest period of the year. This was explained by its high vulnerability to cavitation. The extreme heterogeneity of the six species studied suggested that sclerophylly cannot be interpreted as an anatomical feature leading to a specific and unifying functional role, at least as regards drought resistance.