Structural relatedness between the 18S rRNA genes and the formyl peptide receptor genes: new insights into the phylogenesis of immune receptors.

In this study the authors examined the sequences of the ribosomal 18S rRNA of Drosophila and man and 16 mRNA sequences coding for different members of the family of the mammalian formyl peptide receptors (FPRs). The positions in the sequences of all >or=7-base oligonucleotide identities occurring in at least one of the 18S rRNAs and one of the FPR mRNAs were recorded. On the basis of the positional data, the Drosophila 18S-FPR and human 18S-FPR distances (in nucleotides) were determined for each identity. Then the actual frequency distribution of the distances (grouped into 200-unit classes) was derived. The theoretical frequency distribution of distances was also calculated under the assumption of non-relatedness between the 18S and FPR sequences. Comparison between the theoretical and the actual distributions showed that at class -500 (range from - 400 to - 600) of the 18S-FPR values the actual frequency was significantly (p < 0.01) higher than the theoretical frequency, in both Drosophila and man, suggesting that the second section of the FPR genes (approximately from nucleotide 400 to the end of sequence) may be structurally related to the first section of the ribosomal 18S genes (approximately nucelotides 1-650). The authors advance the hypothesis that the two families of genes may have used common ancestral raw genetic materials in the building of the extant sequences.

The Structure of the 18S rRNA, a molecule that might be evolutionarily related to some receptors of innate immunity.

Comparisons between the sequences of insect and vertebrate 18S rRNAs and the sequences of mammalian formyl peptide and some vertebrate chemokine receptor mRNAs demonstrated non-random structural similarities between these two groups of RNAs. It has been proposed that sections of the more ancient and conserved rRNA genes could have participated in the building of these more recent genes involved in immune responses. Here we analyze the sequence architecture of the 18S rRNA in insects (Drosophila simulans) and vertebrates (man), in terms of similarities between selected segments within the individual molecules. The insect and vertebrate 18S rRNAs are basically similar, but show specific insertions/deletions and base changes. In spite of these differences, in both sequences a significantly higher-than-expected (by random occurrence) number of 7-or-more-base oligonucleotide repeats was observed between segments roughly corresponding to nt 350-1050 and nt 1150-1850, with mutual between-repeats distances comprised in the range 700-900 nt. Based on this result we performed a multialignment of segments 317-1035 of Drosophila, 360-1005 of man, 1096-1864 of Drosophila, and 1066-1736 of man, the first two segments covering the region of first occurrence of the repeats and the last two the region of recurrences. At both ends of these segments the four sequences could be aligned with relatively minor gaps and the number of base identities in all four sequences was significantly higher than expected by random coincidences. These results support the hypothesis that an ancestral gene structure, composed of a chain of about 700 nt, duplicated to form a two-unit tandem repeat which still represents the most substantial part of the 18S rRNA molecule in extant insects and vertebrates.

Evolution of a "conserved" amino acid sequence: a model study of an in silico investigation of the phylogenesis of some immune receptors.

In this paper we analyze a 55-amino acid (aa) sequence which is relatively well conserved in several seven-transmembrane receptor families (from Insects to Mammals) and in some Viruses. This sequence, which covers the second transmembrane domain, the first extracellular loop and the third transmembrane domain, appears in its complete configuration in most of the seven-transmembrane receptor families, as well as in the protein products of some viruses. Other seven-transmembrane receptors and viruses exhibit reduced configurations of the conserved sequence, lacking either aa 31 or aa 30-31. 53-aa configurations are typically found in most chemokine receptor (CKR) subfamilies, as well as in some viral protein products. However, the CCR1, CCR3, and CCR6 subfamilies comprise a 54-aa configuration and the CKR-related protein products, ChemR23 and RDC1, include the complete 55-aa sequence. For each CKR subfamily the "modal sequence" of the conserved segment was constructed by selecting the most frequently occurring aa at each position. Then, pairwise alignments were made between: (i) the modal CKR sequences, and (ii) the sequence (53-aa) of the Yaba-like disease virus - 7L protein. From the alignments two consensus matrices were derived: (i) the consensus 1 matrix with reference to the whole conserved segment, and (ii) the consensus 2 matrix with reference to aa 22-29, which appear to be the most variable segment of the sequence. Based on the obtained consensus values and with reference to this specific conserved segment, the following conclusions are proposed: (1) ChemR23 and RDC1 are probably the more primitive CKR forms; (2) CCR1 and CCR3 may be grouped in a single cluster; (3) CCRs 2, 4, and 5 are closely related to each other and may be grouped in a cluster; CCR7 is likely to be evolutionarily related to this cluster; (4) CXCRs 2, 3, and 4 and CCX CKR appear to be evolutionarily related to each other and very likely derived from an CCR6-like gene; (5) CCR2/4/5 and CCR7 may have derived either from CCR1/3-like or CCR6-like genes; (6). The Yaba-like disease virus--7L protein most likely derived, through "molecular piracy", from a CCR8-like gene. We also discuss possible, more remote, evolutionary links between CKRs, formylpeptide receptors, and possibly the highly conserved 18S rRNA genes.

Polimorphonuclear cell-mediated oxidative stress: sink for reactive oxygen species and cell various type damage.

Reactive oxygen species (ROS) are produced in animals and humans under physiologic and pathologic conditions. Polymorphonuclear cells (PMNs) and other professional phagocytes are able to generate large amounts of ROS that have not only antimicrobial capacity but are also deleterious to mammalian cells and responsible for many chronic diseases. In particular, ROS produced in large amounts by the massively infiltrating leukocytes in inflammed tissues are believed to constitute a major tissue-destructive force and may contribute significantly to the pathogenesis of several inflammatory diseases. Inflammation can accelerate the development of cancer: in fact, it seems that a part of the predisposition to cancer may be attributed to the oxidants released by the phagocytes at inflammatory site and then to the effects of continuous damage over a life span by ROS. The focus of this study was to investigate the differential capacity of ROS capture and the relative cellular damage degree in gastric, intestinal and fibroblastic cell lines. These various cell types were in vitro used as sink for ROS released by co-cultured fMLP-stimulated human polymorphonuclear cells. Our data demonstrated that cell lines showed a differential capacity of ROS capture correlated to cellular damage, probably due to a different cell susceptibilty to the oxidative challenge produced by stimulated PMNs.

Structural similarities between mRNA for the formyl peptide receptors and 18S rRNA.

Formyl peptides released by some bacteria are powerful chemoattractants and activators of mammalian granulocytes and monocytes, acting through 7-transmembrane specific formyl peptide receptors (FPRs). Three distinct segments of the formyl peptide receptor 1 (FPR1) mRNA of Man share probabilistically significant homologies with segments of the 18S rRNA which are highly conserved from Drosophila to Man. Overall, the three segments cover approximately 24% that of the 18S rRNA sequence and approximately 36% of the FPR1 sequence. The three segments are, however, arranged in different orders in the 18S rRNAs and in the FPR1 mRNA, the segment appearing in the first location in the 18S rRNAs is located at the end of the FPR1 mRNA sequence. The hypothesis is advanced that the three "conserved" segments either derive from an ancestral gene that is the forerunner of both the ribosomal 18S genes and the FPR genes or that at some stage of evolution the FPR genes derived, at least in part, from the more ancient ribosomal 18S genes. The extant 18S rRNA sequences exhibit obvious signs of a number of breaks that occurred during evolution, especially in the transition from insects to vertebrates. Some of these events may have resulted in differential rearrangements of segments in the groups of FPR genes and ribosomal 18S genes.

Power spectral analysis of the shape of fMLP-stimulated granulocytes. A tool for the study of cytoskeletal organization under normal and pathological conditions.

fMLP (N-formyl-methionyl-leucyl-phenylalanine) is a powerful activator of granulocytes, eliciting different metabolic responses, such as generation of reactive oxygen species, production of arachidonic acid metabolites, and release of lysosomal enzymes. fMLP determines also a dramatic rearrangement of the actin cytoskeleton; under non-gradient conditions this entails characteristic alterations in cell shape (chemokinesis), while under gradient conditions it is instrumental in promoting cell migration up the gradient (chemotaxis). Here we analyze mathematically the cell contour of fMLP-stimulated human granulocytes stimulated with fMLP under non-gradient conditions, using the methods for study of stochastic series. The cell contours were drawn and divided into 200 segments of equal linear length and the angles between consecutive segments were computed. The derived series of angles were examined for autocorrelations and from the autocorrelation function the power spectrum was calculated. Our results show that the pattern of lamellipodial extensions of the cell membrane is not entirely randomly-designed, but it is partly regulated by deterministic components, as revealed by the presence of statistically significant periodicities. Soon after fMLP stimulation, the power spectrum of the cell contours exhibits a single distinct peak at frequency 0.07, indicating a prevalence of prominent lamellipodia, each one covering in the average 1/15 of the linearized cell contour. Some 30 min after fMLP stimulation the power spectrum becomes flatter (indicating a general decrement of the deterministic component), but still presents one single peak; the latter is shifted to the right (frequency 0.13), indicating the prevalence of less prominent and regular, but more numerous, protrusions, each one covering 1/20 to 1/30 of the cell contour.

Inducible nitric oxide synthase and nitric oxide production in Leishmania infantum-infected human macrophages stimulated with interferon-gamma and bacterial lipopolysaccharide.

Nitric oxide produced by an inducible nitric oxide synthase constitutes one of the main microbicidal mechanisms of murine macrophages and its importance is now being recognized for human macrophages. In this study we evaluated inducible nitric oxide synthase expression, nitric oxide release, and parasitocidal ability of Leishmania infantum-infected monocyte-derived human macrophages. The inducible nitric oxide synthase was detected by immunofluorescence and western blotting and nitric oxide production was measured by the Griess reaction for nitrites. Parasite killing was microscopically evaluated by fluorescent dyes. Experiments were performed on macrophages with or without previous stimulation with recombinant human interferon-gamma and bacterial lipopolysaccharide. Inducible nitric oxide synthase expression and nitric oxide release were higher in Leishmania-infected stimulated macrophages than in uninfected cells or infected cells without previous stimulation. Nitric oxide production and parasitocidal activity against Leishmania infantum were reduced in macrophages treated with the nitric oxide synthase inhibitor L-N(G) monomethylarginine. These results suggest a microbicidal role for nitric oxide in human leishmaniasis, with the possible practical application of immunological or pharmacological regulation of nitric oxide synthesis in the treatment of this infection.

[Histochemical study of rabbit duodenal mucosa carried out using lectins]. / Studio istochimico della mucosa duodenale di coniglio condotto mediante l'impiego di lectine.

The Authors report histochemical findings about rabbit's duodenal mucosa. The present study has been carried out using five different lectins (Peanut Agglutinin (PNA), Dolichos Biflorus Agglutinin (DBA), Wheat Germ Agglutinin (WGA), Soybean Agglutinin (SBA), Ulex Europaeus Agglutinin I (UEA-I). These lectins have been labelled with Horseradish Peroxidase and binding sites have been stained with 3-3' Diaminobenzidine, according to Farragiana et al. The PNA reacted with the glandular cells, while the reaction was negative in the superficial cells. The DBA reacted exclusively with the glandular cells. The superficial and the glandular cells showed strong positive binding sites to the WGA and slight positive binding sites to the SBA. The UEA-I did not react with the epithelial cells. The presence of binding sites for the lectins we have used in the present study, shows a different glycoprotein composition of the cellular secretion, in comparison with the other animals we have already studied. In addition, these lectins can not be used as cellular differentiation markers in the epithelial cells of the rabbit's duodenal mucosa.

[Relation between variance and the arithmetic mean in frequency distributions under conditions of stochastic dependence]. / Rapporti fra varianza e media aritmetica di distribuzioni di frequenza, in condizioni di dipendenza stocastica.

While the average value of the mitotic index in a primordium is determined by genetic and epigenetic factors, the spatial location of the single mitotic events is stochastic; consequently mitotic density is uneven even in homogeneous areas of the primordium. The statistical frequency distribution of the mitotic events has been studied under three different conditions: (a) no stochastic dependence between adjoining events (random distribution); (b) positive stochastic dependence (facilitation) between adjoining events; (c) negative stochastic dependence (inhibition) between adjoining events. It is concluded that in the first case the variance of the distribution equals the arithmetic mean; in the second case the variance is higher than mean; in the third case variance is lower than mean.