Pore formation by the sea anemone cytolysin equinatoxin II in red blood cells and model lipid membranes

Summary The interaction ofActinia equina equinatoxin II (EqT-II) with human red blood cells (HRBC) and with model lipid membranes was studied. It was found that HRBC hemolysis by EqT-II is the result of a colloid-osmotic shock caused by the opening of toxin-induced ionic pores. In fact, hemolysis can be prevented by osmotic protectants of adequate size. The functional radius of the lesion was estimated to be about 1.1 nm. EqT-II increased also the permeability of calcein-loaded lipid vesicles comprised of different phospholipids. The rate of permeabilization rised when sphingomyelin was introduced into the vesicles, but it was also a function of the pH of the medium, optimum activity being between pH 8 and 9; at pH 10 the toxin became markedly less potent. From the dose-dependence of the permeabilization it was inferred that EqT-II increases membrane permeability by forming oligomeric channels comprising several copies of the cytolysin monomer. The existence of such oligomers was directly demonstrated by chemical cross-linking. Addition of EqT-II to one side of a planar lipid membrane (PLM) increases the conductivity of the film in discrete steps of defined amplitude indicating the formation of cation-selective channels. The conductance of the channel is consistent with the estimated size of the lesion formed in HRBC. High pH and sphingomyelin promoted the interaction even in this system. Chemical modification of lysine residues or carboxyl groups of this protein changed the conductance, the ion selectivity and the current-voltage characteristic of the pore, suggesting that both these groups were present in its lumen.     

Hepatic respiratory compensation and haematological changes in the cave cyprinid, Phreatichthys andruzzii

In several ectotherms, including all members of the Osteichthyes studied so far, the spleen is capable of storing and releasing erythrocytes according to the animal's respiratory needs. The tropical cave cyprinid Phreatichthys andruzzii uses its liver rather than the spleen as the site of accumulation in the respiratory compensation process, like the amphibian Rana esculenta. The reversible process of erythrocyte accumulation in the liver is very evident in animals anaesthetized with chlorobutanol; MS-222, an anaesthetic widely used in lower vertebrates alters all the haematological parameters and is not suitable for studies on blood and respiration. The hepatic respiratory compensation mechanism is as efficient as the splenic one: in animals kept at 18 °C for 24 h the mean liver weight percentage was 70% higher than in specimens kept at 28 °C (2.43% of the body weight compared to 1.39%, in groups of six specimens) while mean red blood cell counts fell from 2.49 to 1.60 · 10¹² per l, in agreement with the haematocrit value and haemoglobin concentration; mean corpuscular volume remained constant (at about 177 fl). Accepted: 15 April 1997     

Structural biology: Paper alert

A selection of interesting papers that were published in the two months before our press date in major journals most likely to report significant results in structural biology.     

A novel mechanism of pore formation: membrane penetration by the N-terminal amphipathic region of equinatoxin

Equinatoxin II is a representative of actinoporins, eukaryotic pore-forming toxins from sea anemones. It creates pores in natural and artificial lipid membranes by an association of three or four monomers. Cysteine-scanning mutagenesis was used to study the structure of the N terminus, which is proposed to be crucial in transmembrane pore formation. We provide data for two steps of pore formation: a lipid-bound monomeric intermediate state and a final oligomeric pore. Results show that residues 10-28 are organized as an alpha-helix in both steps. In the first step, the whole region is transferred to a lipid-water interface, laying flat on the membrane. In the pore-forming state, the hydrophilic side of the amphipathic helix lines the pore lumen. The pore has a restriction around Asp-10, according to the permeabilization ratio of ions flowing through pores formed by chemically modified mutants. A general model was introduced to derive the tilt angle of the helix from the ion current data. This study reveals that actinoporins use a unique single helix insertion mechanism for pore formation.     

Behavioral and electrophysiological responses of the parasitic wasp Psyttalia concolor (Szépligeti) (Hymenoptera: Braconidae) to Ceratitis capitata-induced fruit volatiles

Psyttalia concolor (Szépligeti) is a koinobiont larval-pupal endoparasitoid of many Tephritidae of great economic importance, such as the medfly, Ceratitis capitata (Wiedemann). In several species of parasitoids it has been demonstrated that the mated females are strongly attracted by specific volatiles from insect-damaged plants. Yet the role of olfactory cues deriving from C. capitata-infested fruits on the female’s decision during the P. concolor host location was poorly investigated. In the present study, the responses of P. concolor females to either healthy or C. capitata-infested fruits was studied through behavioral assays. Volatiles emitted by healthy and infested fruits were SPME-sampled and analyzed by gas chromatography–mass spectrometry (GC–MS). The attractiveness of the identified volatiles was assessed and their electrophysiological activity was analyzed through gas-chromatography coupled with electroantennography (GC-EAD). P. concolor preferred infested peaches and apples over healthy ones, either when visual and olfactory or only olfactory cues were given. Nine compounds were found as exclusive of infested peaches, with respect to healthy ones, and seven of them evoked electrophysiological responses. In apples only quantitative changes in volatile emissions were observed after the medfly infestation. The emissions of 1-butyl butylate, 1-hexyl acetate and 1-butyl esanoate increased in infested apples, whereas 1-hexyl (E)-2-methyl butenoate decreased significantly. Among apple volatiles, 1-butyl butylate, 2-methyl-1-butyl acetate, 1-hexyl acetate, 2-methyl-1-butyl 2-methylbutanoate, 1-butyl hexanoate and 1-hexyl (E)-2-methyl butenoate elicited responses in female antennae. Synthetic blends reproducing the odors emitted by infested peaches and apples elicited strong attraction towards P. concolor females. For both fruits, the blend attractiveness was mainly due to some specific electrophysiological active chemicals: ethyl octanoate, decanal and 4-decanolide for peach, and 1-butyl butylate and 1-butyl hexanoate for apple. The responses induced by the identified fruit volatiles to P. concolor females allow us to suppose that they play a role as short-range attractants during host location.     

Morphological observations on some fleshy crustose algae of the Island of Gorgona (Tuscan Archipelago)

Abstract

Morphological and ecological observations on four fleshy crustose algae collected in the eulittoral and upper sublittoral zone of the Island of Gorgona: Myrionema liechtenstenii Hauck, Hildenbrandia canariensis B?rgesen, Peyssonnelia armorica (P. et H. Crouan) Weber-van Bosse in B?rgesen and Cruoria cruoriaeformis (P. et H. Crouan) Denizot are reported. For each species, information on some aspects of their ecology, geographical distribution and notes on their vegetative and reproductive morphology are given.     

Use of somatic cell nuclear transfer to study meiosis in female cattle carrying a sex-dependent fertility-impairing X-chromosome abnormality

Animal models have played an important part in establishing our knowledge base on reproduction, development, and the occurrence and impact of chromosome abnormalities. Translocations involving the X chromosome and an autosome are unique in that they elicit sex-dependent infertility, with male carriers rendered sterile by synaptic anomalies during meiosis, whereas female carriers conceive but repeatedly abort. Until now the limited access to relevant fetal oocytes has precluded direct study of meiotic events in female carriers. Because somatic cell nuclear transfer (SCNT) circumvents meiotic problems associated with fertility disturbances in translocation carriers, we used SCNT to generate embryos, fetuses, and calves from a cell line derived from a deceased subfertile X-autosome translocation carrier cow to study the meiotic configurations in carrier oocytes. Data from 33 replicates involving 2470 oocyte-donor-cell complexes were assessed for blastocyst development and of these, 42 blastocysts were transferred to 21 recipients. Fourteen pregnancies were detected on day 35 of gestation. One of these was sacrificed for ovary retrieval on day 94 and three went to term. Features of oocytes from the fetal ovary and from the newborn ovaries were examined. Of the pachytene spreads analyzed, 16%, 82%, and 1.5% exhibited quadrivalent, trivalent/univalent, and bivalent/univalent/univalent structures, respectively, whereas among the diakinesis/metaphase I spreads, 16% ring, 75% chain, and 8.3% bivalent/bivalent configurations were noted, suggesting that the low fertility among female carriers may be related to synaptic errors in a predominant proportion of oocytes. Our results indicate that fibroblasts carrying the X-autosome translocation can be used for SCNT to produce embryos, fetuses, and newborn clones to study such basic aspects of development as meiosis and to generate carriers that cannot easily be reproduced by conventional breeding.     

Kinetic characterization of lipid II-Ala:alanyl-tRNA ligase (MurN) from Streptococcus pneumoniae using semisynthetic aminoacyl-lipid II substrates

MurM and MurN are tRNA-dependent ligases that catalyze the addition of the first (L-Ala/L-Ser) and second (L-Ala) amino acid onto lipid II substrates in the biosynthesis of the peptidoglycan layer of Streptococcus pneumoniae. We have previously characterized the first ligase, MurM (Lloyd, A. J., Gilbey, A. M., Blewett, A. M., De Pascale, G., El Zoeiby, A., Levesque, R. C., Catherwood, A. C., Tomasz, A., Bugg, T. D., Roper, D. I., and Dowson, C. G. (2008) J. Biol. Chem. 283, 6402-6417). In order to characterize the second ligase MurN, we have developed a chemoenzymatic route to prepare the lipid II-Ala and lipid II-Ser substrates. Recombinant MurN enzymes from penicillin-resistant (159) and -sensitive (Pn16) S. pneumoniae were expressed and purified as MBP fusion proteins and reconstituted using a radiochemical assay. MurN ligases from strains 159 and Pn16 both showed a 20-fold higher catalytic efficiency for lipid II-L-Ala over lipid II-l-Ser, with no activity against unmodified lipid II, and similar kinetic parameters were measured for MurN from penicillin-resistant and penicillin-sensitive strains. These results concur with the peptidoglycan analysis of S. pneumoniae, in which the major cross-link observed is L-Ala-L-Ala. The combined action of ligases MurM and MurN is therefore required in order to rationalize the high level of dipeptide cross-links in penicillin-resistant S. pneumoniae, with ligase MurM showing the major difference between penicillin-resistant and penicillin-sensitive strains.     

Effect of age on surface molecules and cytokine expression in human dendritic cells

Dendritic cells (DCs) are central in regulating both innate and acquired immunity, but their possible age-related functional modifications are still unclear. Here we have analyzed the effect of age on LPS-treated monocyte-derived DCs (MDDCs). A negative correlation between age and cell expression of ICAM-1, CD25 and IL-10 was observed in a group of healthy donors. This has been confirmed by a significantly reduced expression of the same molecules in cells of subgrouped elderly versus younger individuals. On the contrary, a positive correlation between age and cell expression of IL-6 and IL-18 has been reported in all the subjects and further supported by a significant increase of the two pro-inflammatory cytokines in cells of elderly versus young subjects.Our data indicate that aging can impair the expression of ICAM-1 and CD25 and skew the production of cytokines, including IL-18, which concur to make a pro-inflammatory milieu. Altogether, the present results point to additional molecules whose role might be relevant in immunosenescence of human DCs, confirming that these cells undergo functional changes during aging.     

Redox proteomics of fat globules unveils broad protein lactosylation and compositional changes in milk samples subjected to various technological procedures

The Maillard reaction between lactose and proteins occurs during thermal treatment of milk and lactosylated β-lactoglobulin, α-lactalbumin and caseins have widely been used to monitor the quality of dairy products. We recently demonstrated that a number of other whey milk proteins essential for nutrient delivery, defense against bacteria/virus and cellular proliferation become lactosylated during milk processing. The extent of their modification is associated with the harshness of product manufacturing. Since fat globule proteins are also highly important for the health-beneficial properties of milk, an evaluation of their lactosylation is crucial for a complete understanding of aliment nutritional characteristics. This is more important when milk is the unique dietary source, as in the infant diet. To this purpose, a sequential proteomic procedure involving an optimized milk fat globule (MFG) preparation/electrophoretic resolution, shot-gun analysis of gel portions for protein identification, selective trapping of lactosylated peptides by phenylboronate chromatography and their analysis by nanoLC-ESI-electron transfer dissociation (ETD) tandem MS was used for systematic characterization of fat globule proteins in milk samples subjected to various manufacturing procedures. Significant MFG protein compositional changes were observed between samples, highlighting the progressive adsorption of caseins and whey proteins on the fat globule surface as result of the technological process used. A significant lactosylation of MFG proteins was observed in ultra-high temperature sterilized and powdered for infant nutrition milk preparations, which well paralleled with the harshness of thermal treatment. Globally, this study allowed the identification of novel 157 non-redundant modification sites and 35 MFG proteins never reported so far as being lactosylated, in addition to the 153 ones ascertained here as present on other 21 MFG-adsorbed proteins whose nature was already characterized. Novel MFG proteins include components involved in nutrient delivery, defense response against pathogens and cellular proliferation/differentiation. Nutritional, biological and toxicological consequences of these findings are here discussed, highlighting their possible impact on children's diet.