Phylogeny and genetic code: evolution and conservation.

After a general view of the actual situation of the phylogenetic relationships between the diverse animals and thus having traced the fundamental elements of an essentially phylogenetic classification, several themes that emerge from this initial analysis have been developed. For instance one of the fundamental problems is about homologous genes shared by the protostomes and deuterostomes and attributed back to that hypothetical group of the primitive bilaterians, the Urbilateria, "reconstructed" from genetic analysis. The analyzed genes are essentially of a regulatory type, thus a remarkable conservation of functions seems to exist. This fundamental conservation of the general genetic patrimony of the living organisms, within an overall picture which includes an infinite gamma of structural and functional modulations of the single genes, and a vast gamma of temporal and spatial modulations of regulation of the genic activity, justifies the use of diverse animal forms for the study and comprehension of general biological phenomena, potentially relevant for human health.

Ultrastructure and functional versatility of hirudinean botryoidal tissue.

In leeches, the botryoidal tissue is composed of two different cell types--granular botryoidal cells and flattened endothelial-like cells--localized in the loose connective tissue between the gut and the body wall sac. We have observed that the botryoidal tissue undergoes functional and structural modifications in response to the different needs arising during the life-cycle of the animal. In healthy, untreated leeches, botryoidal cells are organized in cords or clusters, sometimes surrounding few, small lacunae. Conversely, in wounded animals we have observed the transition of the botryoidal tissue from cluster/cord-like structures to a hollow/tubular architecture, typical of pre-vascular structures. We have documented in botryoidal cell cytoplasm the presence of large calcium storage. Moreover, the cytoplasm of botryoidal cells was filled with granules of different form and size, containing iron or melanin, as tested by classic histochemical methods. The presence of elements like iron and calcium was confirmed by the well-established EDS analysis. In response to a surgical wound, botryoidal tissue cells changed their shape and formed new capillary vessels. Concurrently, botryoidal cells secreted iron from cytoplasmic granules into the new cavity: this secretory activity appeared to be related to intracellular calcium fluctuations. At the end of the angiogenic process, botryoidal cells lost their contact with the basal lamina and moved freely in the circulating fluid towards the lesioned area. Interestingly, circulating botryoidal cells were found to carry melanin in the wounded area. This function is probably involved in defense processes. Thus, we have shown that stimulated botryoidal tissue displays a variety of striking structural, secretory and defensive activities.

Hirudo medicinalis: a new model for testing activators and inhibitors of angiogenesis.

An increasing body of evidence indicates that in the leech Hirudo medicinalis the angiogenic process is finely regulated and coordinated by the botryoidal tissue. In this paper we provide evidence on the involvement of botryoidal tissue cells in angiogenesis induced in H. medicinalis by a variety of stimuli including surgical wounds or the administration of modulators of neovascularization. Interestingly, we show that either human activators of vascular cell growth, or anti-angiogenic peptides like angiostatin and endostatin, or the drug mitomycin, can induce a prompt biological response in H. medicinalis. We show as well that angiogenesis in this invertebrate shares a surprising degree of similarity with neovascularization in vertebrates, both at the biochemical and cellular levels, because it involves similar growth factors/growth factor receptors, and relies on analogous cell-cell or cell-matrix interactions. For these reasons we suggest that H. medicinalis can be used as a reproducible model for testing activators or inhibitors of angiogenesis, and for investigating the biochemical, ultrastructural and cellular processes involved in new vessel formation.

Different types of response to foreign antigens by leech leukocytes.

We used morphological and immunocytochemical approaches to characterize and to show the behavior of cells involved in leech inflammatory responses. Leeches were injected with bacterial lipopolysaccharide, fluoresceinated yeasts, sulfate spheres and ciliates (Protozoa). Shortly after injection, migrating cells appeared in the area of injection. The response of the cells occurred in relation to the injected micro or macro antigens. Each injection first provoked a migration of cells towards the non-self material. Afterwards, different responses (degranulation, phagocytosis, encapsulation, melanization) occurred. The migrating cells involved in these series of processes have a similar behavior and are characterized by CD markers of macrophages, NK cells and granulocytes, which are typical of many invertebrates and vertebrates.

Structural and biochemical analysis of the parasite Gordius villoti (Nematomorpha, Gordiacea) cuticle.

The cuticle of the nematomorpha Gordius villoti is a proteinaceous extracellular structure that covers the body during the endoparasitic life in the hemocoelic cavity of insect hosts, and of the free-living adult animals. The ultrastructure of the cuticle has a complex spatial organization with several parallel layers of large diameter fibers, interposed thinner fibrous elements and honeycomb-shaped matrix surrounding the fibers. When adult isolated cuticles were partially solubilized by several compounds, the structure revealed a strong insolubility and the main fibers were always observable. HPLC and spectrophotometric assays carried out to investigate the presence of tyrosine cross-linking, indicated such a mechanism as a key-element in the hardening process of the cuticle. Such data strongly suggest that the Gordius cuticle contains dityrosine compounds, whose formation is probably mediated by endogenous peroxidase activity.

Integumental amino acid uptake in a carnivorous predator mollusc (Sepia officinalis, Cephalopoda).

The epithelial cells of the integument of body, arms and tentacles of Sepia officinalis present on their apical membrane a well-organised brush border and show the morphological and histochemical characteristics of a typical absorptive epithelium. The ability of the integument to absorb amino acids was investigated both in the arms incubated in vitro and in a purified preparation of brush border membrane vesicles (BBMV). Autoradiographic pictures of the integument after incubation of the arms in sea-water with or without sodium, showed that proline intake was Na+-dependent, whereas leucine intake appeared to be a largely cation-independent process. Time course experiments of labelled leucine, proline and lysine uptakes in BBMV evidenced that these amino acids are accumulated within the vesicles in the presence of an inwardly directed sodium gradient. The sodium-driven accumulation proves that cationic and neutral amino acids are taken up by the apical membrane of the epithelium of Sepia integument through a secondary active mechanism. For leucine, a 90% inhibition of the uptake was recorded in the presence of a large excess of the substrate. In agreement with the autoradiography results, an analysis of the cation specificity transport in BBMV showed that leucine uptake had a low cation specificity, whereas lysine and proline uptakes were Na+-dependent. An excess of lysine and proline, which share with alanine two different transport systems in the gill epithelium of marine bivalves, reduced eucine uptake. The possible role of the absorptive ability of the integument in a carnivorous mollusc is discussed.

Lipopolysaccharide-dependent induction of leech leukocytes that cross-react with vertebrate cellular differentiation markers.

We have designed experiments to characterise leech leukocytes that mediate inflammatory responses. Shortly after inflicting injury to the body wall in the presence of lipopolysaccharides, many cells resembling macrophages, NK cells and granulocytes of vertebrates and many invertebrates migrated to the lesioned area. Nuclei of migrating cells incorporated bromodeoxyuridine. Using human monoclonal antibodies, macrophage-like cells were positive for CD25, CD14, CD61, CD68, CD11b and CD11c. NK-like cells were positive for CD25, CD56, CD57 and CD16, and granulocytes were positive for CD11b and CD11c. In blots of leech extracts, the CD25 monoclonal antibody recognised a band of about 55 kD; the CD56 monoclonal antibody, two bands of about 140 and 210 kD; the CD57 monoclonal antibody, two bands of about 106 and 70 kD; the CD14 monoclonal antibody, a band of about 50 kD; the CD16 monoclonal antibody, a band of about 60 kD. CD61 and CD68 both recognised a band of about 110 kD; CD11b recognised a band of 200 kD, and CD11c, a band of 180 kD.

Possible roles of extracellular matrix and cytoskeleton in leech body wall muscles.

Round circomyarian fibres of leeches are peculiar helical muscles. The fibres are characterized by a lack of junctions, being separated by a thick extracellular matrix, and by scarce end-plates. Even so, the fibres grouped in units show the same degree of contraction. Biochemical, immunocytochemical and ultrastructural studies were performed in order: (a) to demonstrate the presence in the extracellular matrix of fibronectin, collagen type IV and laminin and in the cytoskeleton of desmin and alpha-actinin; (b) to show the possible link of extracellular matrix with the scaffold of intermediate filaments; (c) to evaluate how the extracellular matrix can play a role in the transduction of a signal during contraction-relaxation-superelongation phases.

Histopathological changes after induced injury in leeches.

Cells involved in leech inflammatory responses have been characterized by morphological, histochemical, and immunohistochemical methods. Macrophage-like cells, NK-like cells, and granulocytes migrated shortly after injury by pricking with bacterial lipopolysaccharide. Inflammatory responses increased progressively and provoked cell migration to the body wall and then to wound surfaces. Macrophages, NK cells, and granulocytes display similar features and behavior traits in invertebrates and vertebrates.

Trophic exchanges between parent and young during development of Glossiphonia complanata (Annelida, Hirudinea).

Glossiphoniids are iteroparous hirudineans that brood their offspring. The young are attached by the posterior sucker to the parent's ventral side until development is complete. The weight loss of the brooding adults is commonly attributed to their reduced possibility of feeding and to the increased metabolic expense for movements needed to ventilate the offspring. The present study showed that there is a passage of nutritive substrates between the parental body wall and the young's sucker. A possible correlation between this passage and the reabsorption of some muscle fibers that occurs in the adult's body wall is also analyzed.

Analysis and reconstruction of unusual obliquely striated fibres in Lumbriculids (Annelida, Oligochaeta).

The longitudinal muscle fibres of Lumbriculids (Annelida, Oligochaeta) have been studied by means of electron microscopy. These fibres are obliquely striated (flattened circomyarian) and possess Z-elements and sarcotubules which frequently cross the centre of the fibre connecting the cell surface on the opposite sides. The myosin filaments have, in cross-section, a rectangular profile with the long side parallel to the fibre surface; they look wavy (S-shaped) on the XZ plane, thus crossing the whole thickness of the sarcomere. The three-dimensional interpretation of the contractile apparatus was facilitated by the comparison of the micrographs from ad hoc sections of the three spatial planes with corresponding calculated "sections', obtained with a dynamic computer model. The real periodicity of the different contractile structures has been determined and the wavy shape of the thick filaments on the XZ plane has been demonstrated by indirect proofs: computed images in which rectangular filaments are parallel to the fibre axis cannot be made to yield a situation comparable with real images. Furthermore, the distances (centre to centre) between thick filaments in fibres at different states of contraction are so distributed in each "sarcomere' as to be compatible only with the wavy filaments.

Superelongation in helical muscles of leeches.

The fine structure of leech body wall muscles, has been analysed under great length variations. All the measurable parameters of the fibre (distance between thick filaments, sarcomere and A band width, percentage of actin crowns around myosin filaments, fibre radius) at different elongations were tested with equations describing the geometrical model of helical fibres. Anaesthetized and nonanaesthetized worms behave in different ways and can be utilized to verify the 'changing partner hypothesis'. The fit between theoretical and experimental data suggests that a double change of partner actually takes place in extremely stretched fibres.

Isolation and identification of paramyosin from amphioxus notochord.

Paramyosin was extracted and purified from the notochordal lamellae of the amphioxus, Branchiostoma lanceolatum. The subunit molecular weight, as determined by dodecyl sulphate gel electrophoresis, was 108 000. The amino acid composition of amphioxus paramyosin, although similar to that of invertebrate paramyosin, showed differences: a higher content of proline, a lower amount of dicarboxylic amino acids, and a lysine/arginine ratio greater than one. Electron microscopy of paracrystals formed by divalent cations showed the axial periodicities of 144 or 725 A and the subperiod of 48 A, which is specific for paramyosin. The fine structure of amphioxus paracrystals differed from that of molluscan paramyosin.

Discontinuity of sarcoplasmic reticulum in the mid-sarcomere region in flight muscle of dragonflies.

The sarcoplasmic reticulum organization of dragonfly flight muscles is analyzed, with particular reference to the doubling existing at H-band level. This doubling could be explained as a consequence of a regular discontinuity in the sarcoplasmic reticulum covering myofibrils. In each sarcomere, two sleeves of the sarcoplasmic reticulum seem to overlap forming a telescopic system which can slide outside each other during the lengthening and shortening movements of the fiber.

Flight muscle differentiation in nymphs of a dragonfly Anax imperator.

Flight muscle fibers of Anax imperator nymphs, in different developmental stages are analyzed for several morphological features, such as the arragnement and numerical ratio of actin and myosin filaments, the pattern of the T system and sarcoplasmic reticulum, the number of microtubules and the fractional volume of mitochondria in each fiber. The T system is initially represented by longitudinal grooves on the cell surface, joined with vesicles of the sarcoplasmic reticulum; this pattern rapidly changes and the grooves start to break up into longitudinal segments. The thin to thick filament ratio is at first quite high (about 4-4.5:1) but rapidly falls to the final (3:1) when the myofibrils are well developed at the fiber periphery. Statistical analyses show that the measured values are significantly different in the various stages of development, also indicating a progressive reduction of the ratio variability. The reduction of thin to thick filament ratio and the variance decrease fit quite well with the hypothesis that the synthesis of actin and myosin depends on independently regulated messenger RNA molecules.