Spontaneous Cell Fusion in Macrophage Cultures Expressing High Levels of the P2Z/P2X7 Receptor

Mouse and human macrophages express a plasma membrane receptor for extracellular ATP named P2Z/P2X₇. This molecule, recently cloned, is endowed with the intriguing property of forming an aqueous pore that allows transmembrane fluxes of hydrophylic molecules of molecular weight below 900. The physiological function of this receptor is unknown. In a previous study we reported experiments suggesting that the P2Z/P2X₇ receptor is involved in the formation of macrophage-derived multinucleated giant cells (MGCs; Falzoni, S., M. Munerati, D. Ferrari, S. Spisani, S. Moretti, and F. Di Virgilio. 1995. J. Clin. Invest. 95:1207– 1216). We have selected several clones of mouse J774 macrophages that are characterized by either high or low expression of the P2Z/P2X₇ receptor and named these clones P2Zhyper or P2Zhypo, respectively. P2Zhyper, but not P2Zhypo, cells grown to confluence in culture spontaneously fuse to form MGCs. As previously shown for human macrophages, fusion is inhibited by the P2Z/P2X₇ blocker oxidized ATP. MGCs die shortly after fusion through a dramatic process of cytoplasmic sepimentation followed by fragmentation. These observations support our previous hypothesis that the P2Z/P2X₇ receptor is involved in macrophage fusion.     

Evolution of the Mitochondrial Cytochrome Oxidase II Gene in Collembola

The sequence of the mitochondrial COII gene has been widely used to estimate phylogenetic relationships at different taxomonic levels across insects. We investigated the molecular evolution of the COII gene and its usefulness for reconstructing phylogenetic relationships within and among four collembolan families. The collembolan COII gene showed the lowest A + T content of all insects so far examined, confirming that the well-known A + T bias in insect mitochondrial genes tends to increase from the basal to apical orders. Fifty-seven percent of all nucleotide positions were variable and most of the third codon positions appeared free to vary. Values of genetic distance between congeneric species and between families were remarkably high; in some cases the latter were higher than divergence values between other orders of insects. The remarkably high divergence levels observed here provide evidence that collembolan taxa are quite old; divergence levels among collembolan families equaled or exceeded divergences among pterygote insect orders. Once the saturated third-codon positions (which violated stationarity of base frequencies) were removed, the COII sequences contained phylogenetic information, but the extent of that information was overestimated by parsimony methods relative to likelihood methods. In the phylogenetic analysis, consistent statistical support was obtained for the monophyly of all four genera examined, but relationships among genera/families were not well supported. Within the genus Orchesella, relationships were well resolved and agreed with allozyme data. Within the genus Isotomurus, although three pairs of populations were consistently identified, these appeared to have arisen in a burst of evolution from an earlier ancestor. Isotomurus italicus always appeared as basal and I. palustris appeared to harbor a cryptic species, corroborating allozyme data. Received: 12 January 1996 / Accepted: 10 August 1996     

Long-term gene delivery into the livers of immunocompetent mice with E1/E4-defective adenoviruses.

We have compared the in vitro and in vivo behaviors of a set of isogenic E1- and E1/E4-defective adenoviruses expressing the lacZ gene of Escherichia coli from the Rous sarcoma virus long terminal repeat. Infection of tumor-derived established cell lines of human origin with the doubly defective adenoviruses resulted in (i) a lower replication of the viral backbone that correlated with reduced levels of E2A-specific RNA and protein, (ii) a significant shutoff of late gene and protein expression, and (iii) no apparent virus-induced cytotoxicity. Independently of the extent of the deletion, the additional inactivation of E4 from the viral backbone therefore drastically disabled the virus in vitro, with no apparent effect on transgene expression. A lacZ-transgenic model was used to compare the different recombinant adenoviruses in the livers of C57BL/6 mice. The immune response to the virally encoded beta-galactosidase was minimal in this model, as infusion of the E1-defective adenovirus resulted in a time course of transgene expression that mimicked that in immunodeficient (nu/nu) mice, with very little inflammation and necrosis in the liver. Administration of a doubly defective adenovirus to the transgenic animals led to long-term extrachromosomal persistence of viral DNA in the liver, with no detectable methylation of CpG dinucleotides. However, transient transgene expression was observed independently of the extent of the E4 deletion, suggesting that the choice of the promoter may be critical to maintain transgene expression from these attenuated adenovirus vectors.     

Genetic differentiation and detection of cryptic species in the genus Lepismachilis (Insecta: Microcoryphia) from the Western Mediterranean region

Different species of the bristletail genus Lepismachilis were collected in 14 localities in Italy and Spain and an allozyme electrophoretic survey was carried out to estimate the degree of genetic variability and differentiation at intra- and interspecific levels. Four morphological species were initially identified (L osellai, L. y-signata, L. affinis, L. targionii), but the electrophoretic analysis demonstrated the presence of two additional species among the individuals of L. targionii (Lepismachilis spl and sp2). The validity of these species and their differentiation from L targionii were demonstrated by the fixation of alternative allelic patterns at several loci (7 in Lepismachilis spl and 8 in Lepismachilis sp2), coupled with fixed, previously undetected, morphological differences. In addition, Lepismachilis sp2 was sympatric with L. targionii in three collecting sites, where the fixation of alternative allelic patterns unequivocally demonstrated reproductive isolation. Genetic variability did not seem to be correlated with local ecological factors, and differences between species should rather be explained by different historical factors. Low levels of gene flow, estimated with two different indirect methods, were observed in L. targionii and L. y-signata, and were due to high levels of structuring among populations. Genetic differentiation among conspecific populations was not correlated to their geographical arrangement and the presence of loci fixed for different alleles among them suggested that stochastic factors (such as genetic drift) may have played a role in determining genetic differentiation of geographically isolated populations. Genetic divergence values indicated that the six species are well differentiated and allozyme profiles were diagnostic for all of them. On the other hand, allozyme data did not provide adequate information to resolve evolutionary relationships among the species, nor did they confirm the validity of the two subgenera (Lepismachilis and Berlesilis) in which the genus Lepismachilis is traditionally divided.     

Time linkages between pollination onsets of different taxa over an 11-year period in Perugia,Central Italy

Times of pollination of different taxa in the atmosphere of Perugia (Central Italy) over an 11-year period (1982–1992) were recorded and analysed by means of a 7-day volumetric Hirst-type pollen trap. For some taxa, the pollination period varied from year to year from a chronological and/or quantitative point of view. Several taxa showed a linkage in their starting dates of pollination. Knowledge of this kind of linkage allows us to build a forecasting model.     

The role of physical cues in the regulation of host recognition and acceptance behavior ofAphidius ervi Haliday (Hymenoptera: Braconidae)

The role of color and shape in the host recognition and acceptance behavior ofAphidius ervi Haliday was studied. A quantitative analysis of the oviposition behavior ofA. ervi was carried out with a computer-aided analysis of 150 video-recorded oviposition sequences on its natural host,Acyrthosiphon pisum (Harris). The importance of visual stimuli was assessed in a choice condition bioassay, observing the behavioral reaction of female parasitoids to various test materials flame-sealed into glass capillaries. Glass beads 2 and 6 mm in diameter and a flat arena were coated with cornicle secretion ofA. pisum, and their acceptance rates by both naive and experienced female parasitoids were assessed under no-choice conditions. In most cases,A. ervi females switched from random searching to attack position when the host was within a range of 1 cm, suggesting that host recognition is regulated in part by cues acting before physical contact. The glass capillary bioassay indicated that visual cues are important factors in the host recognition and acceptance phases. Pea aphid color alone can elicit the oviposition response of naiveA. ervi females, and this response is enhanced when color is combined with aphid shape. The cornicle secretion ofA. pisum stimulated an oviposition response which was stronger in naive females ofA. ervi than in experienced ones and was not significantly affected by the glass bead size or flat surface. These results, along with those from previous studies, suggest that manipulation of the oviposition behavior ofA. ervi is feasible under laboratory conditions.     

A chlorinated phenylpyrrole antibiotic fromActinoplanes

Two chlorophenyl-containing antibiotics have been isolated from a strain ofActinoplanes (ATCC 33002). Antibiotic A 15104 Y is a chlorinated phenylpyrrole compound whose structure has been confirmed by chemical synthesis. Antibiotic A 15104 Z is a chlorophenol derivative for which a structural formula is proposed on the basis of its physicochemical properties. A 15104 Y shows antimicrobial activity against Gram-positive, Gram-negative, and acid-fast bacteria, yeasts, fungi, and protozoa, while A 15104 Z possesses a low activity against Gram-positive bacteria andTrichophyton. A 15104 Y has a weak activity in curing experimentally infected mice, at a dose that is one-fifth the LD₅₀ for the same species. This is the first example of a chloropyrrole derivative isolated from an actinomycete.     

Freeze-preservation of rice cells: A physiological study of freeze-thawed cells

Rice ( Oryza sativa L.) cells returning to in vitro culture after preservation at superlow temperature in liquid nitrogen are characterized by a number of physiological alterations. These include: reduction in respiration and glucose uptake, loss of intracellular potassium, decrease in the cellular level of key metabolites (ATP, glucose-6-phosphate and pyruvate) and fragility of protoplasts following the action of cell wall-degrading enzymes.
Nevertheless, cell growth resumes after a short lag phase (2–4 days) with an actual 70–100% cell survival, thus indicating that the observed damage is not lethal and can be repaired in a short time.     

The molecular basis of disorders of human hemoglobin synthesis

Summary The structure and organization of the human globin genes at the nucleotide level has been established by restriction endonuclease digestion of cellular DNA, and by the isolation and purification of these. genes in phage vectors. With this approach it has been possible to define alterations at the DNA level resulting in a group of inherited diseases of man known as the thalassemia syndromes, and related disorders. Combined with other known genetic and biochemical data, these studies provide a framework for understanding the pathogenesis of these disorders at the molecular level.     

Sterols from three Lactarius species

The sterol composition of three fungi was determined. Ergosterol is the major sterol, accompanied by other closely related sterols.