Biomedical subjects

G Tettamanti

Publications and source records attributed to G Tettamanti.

At least 19 recordsLinked to original sources

PubMed2006

Structure and function of the extraembryonic membrane persisting around the larvae of the parasitoid Toxoneuron nigriceps.

The embryo of Toxoneuron nigriceps (Hymenoptera, Braconidae) is surrounded by an extraembryonic membrane, which, at hatching, releases teratocytes and gives rise to a cell layer embedding the body of the 1st instar larva. This cell layer was studied at different developmental times, from soon after hatching up to the first larval moult, in order to elucidate its ultrastructural, immunocytochemical and physiological function. The persisting "larval serosa" shows a striking structural and functional complexity: it is a multifunctional barrier with protective properties, limits the passage of macromolecules and it is actively involved in the enzymatic processing and uptake of nutrients. The reported results emphasizes the important role that the embryo-derived host regulation factors may have in parasitism success in Hymenoptera koinobionts.

Animal Nutritional Physiological Phenomena↗

PubMed2006

Growth factors and chemokines: a comparative functional approach between invertebrates and vertebrates.

Growth factors and cytokines control and coordinate a broad spectrum of fundamental cellular functions, and are evolutionarily conserved both in vertebrates and invertebrates. In this review, we focus our attention on the functional phylogenetic aspects of growth factors/cytokines like the Transforming Growth Factor-beta (TGF-beta), the Connective Tissue Growth Factor (CTGF), and the Vascular Endothelial Growth Factor (VEGF). We will also delve into the activites of two chemokine families, interleukin (IL)-8 (or CXCL8) and CC chemokine ligand 2/monocyte chemoattractant protein-1 (CCL2). These molecules have been selected for their involvement in immune responses and wound healing processes, where they mediate and finely regulate various regeneration processes like angiogenesis or fibroplasia, not only in vertebrates, but also in invertebrates.

Animals↗

PubMed2006

Hematopoietic cell formation in leech wound healing.

The angiogenic process in vertebrates and hirudineans has been compared. The leech Hirudo medicinalis, subjected to an angiogenic stimulus (surgical explant or cytokine treatment) responds, as a vertebrate, with the formation of an extensive network of new vessels accompanied by the production of circulating cells. The reviewed data confirm the surprising similarity between hirudinean and vertebrate processes in wound healing, and suggest that basic common events such as antigenic expressions of endothelial and hemopoietic cells, cytokine secretion and regulation as well as extracellular matrix interactions, are conserved and extended across diverse species, tissues and developmental phases.

Animals↗

PubMed2004

Molecular cloning and characterization of NEU4, the fourth member of the human sialidase gene family.

Several mammalian sialidases have been cloned so far and here we describe the identification and expression of a new member of the human sialidase gene family. The NEU4 gene, identified by searching sequence databases for entries showing homologies to the human cytosolic sialidase NEU2, maps in 2q37 and encodes a 484-residue protein. The polypeptide contains all the typical sialidase amino acid motifs and, apart from an amino acid stretch that appears unique among mammalian sialidases, shows a high degree of homology for NEU2 and the plasma membrane-associated (NEU3) sialidases. RNA dot-blot analysis showed a low but wide expression pattern, with the highest level in liver. Transient transfection in COS7 cells allowed the detection of a sialidase activity toward the artificial substrate 4MU-NeuAc in the acidic range of pH. Immunofluorescence staining and Western blot analysis demonstrated the association of NEU4 with the inner cell membranes.

Amino Acid Sequence↗

PubMed2004

Ganglioside/glycosphingolipid turnover: new concepts.

In this review focus is given to the metabolic turnover of gangliosides/glycosphingolipids. The metabolism and accompanying intracellular trafficking of gangliosides/glycosphingolipids is illustrated with particular attention to the following events: (a) the de novo biosynthesis in the endoplasmic reticulum and Golgi apparatus, followed by vesicular sorting to the plasma membrane; (b) the enzyme-assisted chemical modifications occurring at the plasma membrane level; (c) the internalization via endocytosis and recycling to the plasma membrane; (d) the direct glycosylations taking place after sorting from endosomes to the Golgi apparatus; (e) the degradation at the late endosomal/lysosomal level with formation of fragments of sugar (glucose, galactose, hexosamine, sialic acid) and lipid (ceramide, sphingosine, fatty acid) nature; (f) the metabolic recycling of these fragments for biosynthetic purposes (salvage pathways); and (g) further degradation of fragments to waste products. Noteworthy, the correct course of ganglioside/glycosphingolipid metabolism requires the presence of the vimentin intracellular filament net work, likely to assist intracellular transport of sphingoid molecules. ut of the above events those that can be quantitatively evaluated with acceptable reliability are the processes of de novo biosynthesis, metabolic salvage and direct glycosylation. Depending on the cultured cells employed, the percentage of distribution of de novo biosynthesis, salvage pathways, and direct glycosylation, over total metabolism were reported to be: 35% (range: 10-90%) for de novo biosynthesis, 7% (range: 5-10%) for direct glycosylation, and 58% (range: 10-90%) for salvage pathways. The attempts made to calculate the half-life of overall ganglioside turnover provided data of unsure reliability, especially because in many studies salvage pathways were not taken into consideration. The values of half-life range from 2 to 6.5 h to 3 days depending on the cells used. Available evidence for changes of ganglioside/glycosphingolipid turnover, due to extracellular stimuli, is also considered and discussed.

Animals↗

PubMed2003

Vascular endothelial growth factor is involved in neoangiogenesis in Hirudo medicinalis (Annelida, Hirudinea).

Vascular endothelial growth factor (VEGF) is fundamental in vertebrates for correct development of blood vessels. However, there are only few data about the presence of VEGF in invertebrates. In this study the role of VEGF in neovessel formation is investigated in Hirudo medicinalis. The leech is able to respond to administration of human VEGF by formation of new vessels. The response of H. medicinalis to this growth factor is explained by the presence of two specific VEGF-like receptors (Flt-1/VEGFR-1 and Flk-1/VEGFR-2) as demonstrated by immunohistochemistry and biochemical analysis. The VEGF-like produced by this annelid following surgical stimulation determines not only blood vessel formation, proliferation of vascular endothelial cells but also an increase of cytoplasmic calcium levels. The administration of specific VEGF receptor antibodies can inhibit angiogenesis in leeches previously stimulated with VEGF.

Animals↗

PubMed2001

GM3 ganglioside inhibits endothelin-1-mediated signal transduction in C6 glioma cells.

We found that sparse and confluent C6 glioma cells differ both in GM3 content, which increases with cell density, and in endothelin-1 (ET-1)-induced phosphoinositide hydrolysis, which was markedly higher in the sparse cells than in the confluent. Also after manipulation of the cellular GM3 content through treatment with exogenous GM3 or with drugs known to affect GM3 metabolism, the ET-1 effect was inversely related to GM3 cellular levels. Cell treatment with an anti-GM3 mAb resulted in the enhancement of ET-1-induced phospholipase C activation and restored the capacity of GM3-treated cells to respond to ET-1. These findings suggest that the GM3 ganglioside represents a physiological modulator of ET-1 signaling in glial cells.

Animals↗

PubMed2001

Nitric oxide production in living neurons is modulated by sphingosine: a fluorescence microscopy study.

An investigation was carried out into the possible effect of sphingosine (Sph) on nitric oxide (NO) production in living neurons. Differentiated granule cells were used in a dynamic videoimaging analysis of single cells labeled, simultaneously, with FURA-2 and the NO indicator 4,5-diaminofluorescein. The results demonstrate that Sph exerts a potent inhibitory effect on the Ca2+-dependent production of NO, without modifying the [Ca2+]i. The effect appears to be specific as neither ceramide nor Sph-1-phosphate had any effect on the NO and [Ca2+]i levels. The data demonstrate that Ca2+-dependent NO production is a specific Sph target in living granule cells, suggesting that this bioactive sphingoid plays a relevant role in neuronal NO signaling.

Animals↗

PubMed2001

Changes in the lipid turnover, composition, and organization, as sphingolipid-enriched membrane domains, in rat cerebellar granule cells developing in vitro.

In the present paper, we report on the properties of sphingolipid-enriched domains of rat cerebellar granule cells in culture at different stages of neuronal development. The major lipid components of these domains were glycerophospholipids and cholesterol. Glycerophospholipids were 45-75% and cholesterol 15-45% of total lipids of the domains. This corresponded to 5-17% of total cell glycerophospholipids and 15-45% of total cell cholesterol. Phosphatidylcholine, mainly dipalmitoylphosphatidylcholine, was 66-85% of all the glycerophospholipids associated with these domains. Consequently, the palmitoyl residue was significantly enriched in the domains. The surface occupied by these structures increased during development. 40-70% of cell sphingolipids segregated in sphingolipid-enriched membrane domains, with the maximum ganglioside density in fully differentiated neurons. A high content of ceramide was found in the domains of aging neurons. Then, the sphingolipid/glycerophospholipid molar ratio was more than doubled during the initial stage of development, whereas the cholesterol/glycerophospholipid molar ratio gradually decreased during in vitro differentiation. Phosphorylated phosphoinositides, which were scant in the domains of undifferentiated cells, dramatically increased during differentiation and aging in culture. Proteins were minor components of the domains (0.1-2.8% of all domain components). Phosphotyrosine-containing proteins were selectively recovered in the sphingolipid-enriched domain. Among these, Src family protein-tyrosine kinases, known to participate to the process of neuronal differentiation, were associated with the sphingolipid-enriched domains in a way specific for the type of kinase and for the developmental stage of the cell. Proteins belonging to other signaling pathways, such as phosphoinositide 3-kinase and its downstream target, Akt, were not associated with the domains.

Animals↗

PubMed2001

Basic fibroblast growth factor-induced proliferation of primary astrocytes. evidence for the involvement of sphingomyelin biosynthesis.

We recently reported that the marked decrease in cellular ceramide in primary astrocytes is an early event associated with the mitogenic activity of basic fibroblast growth factor (bFGF) (Riboni, L., Viani, P., Bassi, R., Stabieini, A., and Tettamanti, G. (2000) GLIA 32, 137-145). Here we show that a rapid activation of sphingomyelin biosynthesis appears to be the major mechanism responsible for the fall in ceramide levels induced by bFGF. When quiescent astrocytes were treated with bFGF, an increased amount of newly synthesized ceramide (from either l-[(3)H]serine or [(3)H]sphingosine) was directed toward the biosynthesis of sphingomyelin. Conversely, bFGF did not appear to affect ceramide levels by other metabolic pathways involved in ceramide turnover such as sphingomyelin degradation and ceramide biosynthesis, degradation, and glucosylation. Enzymatic studies demonstrating a relevant and rapid increase in sphingomyelin synthase activity after bFGF treatment have provided a convincing explanation for the activation of sphingomyelin biosynthesis. The bFGF-induced increase in sphingomyelin synthase appears to depend on a post-translational activation mechanism. Moreover, in the presence of brefeldin A, the activation of sphingomyelin biosynthesis was abolished, suggesting that the enzyme is located in a compartment other than the Golgi apparatus. Also the phosphatidylcholine-specific phospholipase C inhibitor D609 exerted a potent inhibitory effect on sphingomyelin biosynthesis. Finally, we demonstrate that inhibition of sphingomyelin biosynthesis by brefeldin A or D609 led to a significant inhibition of bFGF-stimulated mitogenesis. All this supports that, in primary astrocytes, the early activation of sphingomyelin synthase is involved in the bFGF signaling pathway leading to proliferation.

Animals↗

PubMed2001

Different behavior of ghost-linked acidic and neutral sialidases during human erythrocyte ageing.

Acidic and neutral sialidases (pH optimum 4.7 and 7.2, respectively) were assayed on human circulating erythrocytes during ageing. The assays were performed on intact erythrocytes and resealed erythrocyte ghost membranes. From young to senescent erythrocytes the acidic sialidase featured a 2.7-fold and 2.5-fold decrease in specific activity when measured on intact cells or resealed ghost membranes, whereas the neutral sialidase a 5-fold and 7-fold increase, respectively. The Ca2+-loading procedure was employed to mimic the vesiculation process occurring during erythrocyte ageing. Under these conditions the released vesicles displayed an elevated content of acidic sialidase, almost completely linked through a glycan phosphoinositide (GPI) anchor but no neutral sialidase activity, that was completely retained by remnant erythrocytes together with almost all the starting content of sialoglycoconjugates. The loss with vesiculation of acidic sialidase with a concomitant relative increase of neutral sialidase was more marked in young than senescent erythrocytes. The data presented suggest that during ageing erythrocytes loose acidic sialidase, and get enriched in the neutral enzyme, the vesiculation process, possibly involving GPI-anchors-rich membrane microdomains, being likely responsible for these changes. The enhanced neutral sialidase activity might account for the sialic acid loss occurring during erythrocyte ageing.

Adult↗

PubMed2001

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.

Angiogenesis Inhibitors↗

PubMed2001

Immunoseparation of sphingolipid-enriched membrane domains enriched in Src family protein tyrosine kinases and in the neuronal adhesion molecule TAG-1 by anti-GD3 ganglioside monoclonal antibody.

Rat cerebellar granule cells differentiated in culture were fed [1-(3)H]sphingosine, allowing the metabolic radiolabelling of all cell sphingolipids and phosphatidylethanolamine. A detergent-insoluble sphingolipid-enriched membrane fraction, containing about 60% of cell sphingolipids, but only trace amounts of phosphatidylethanolamine, was prepared from [1-(3)H]sphingosine-fed cells by sucrose gradient centrifugation. This fraction was enriched in the Src family protein tyrosine kinases c-Src, Lyn and Fyn and in the GPI-anchored neuronal adhesion molecule TAG-1. The cell lysate and the sphingolipid-enriched membrane fraction were subjected to immunoprecipitation with anti-GD3 ganglioside monoclonal antibody R24, under experimental conditions designed to preserve the integrity of the domain. The radioactive lipid composition of the immunoprecipitates obtained from the cell lysate and from the sphingolipid-enriched fraction were very similar, and closely resembled the sphingolipid composition of the whole sphingolipid-enriched membrane fraction. In fact, the immunoprecipitates contained, together with GD3 ganglioside, all cell glycosphingolipids and sphingomyelin, whereas they did not contain phosphatidylethanolamine. Moreover, cholesterol and phosphatidylcholine were detected in the immunoprecipitates by qualitative TLC analysis followed by colourimetric visualization. c-Src, Lyn, Fyn and TAG-1 were associated with the anti-GD3 antibody immunoprecipitate. These proteins were not detected in the immunoprecipitates obtained under experimental conditions different from those designed to preserve the integrity of the domain. These data suggest that a membrane domain containing cholesterol, phosphatidylcholine, sphingolipids and proteins can be separated from the total cell membranes by anti-GD3 antibody immunoprecipitation, and that the association of c-Src, Fyn, Lyn, and TAG-1 with the sphingolipid-enriched domain is mediated by the interaction with a complex lipid environment, rather than by specific interactions with a single sphingolipid species.

Animals↗

PubMed2001

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.

Animals↗

PubMed2001

Casein phosphopeptides influence calcium uptake by cultured human intestinal HT-29 tumor cells.

We investigated the direct effects of casein phosphopeptides (CPP), which are formed by the proteolytic degradation of alpha- and beta-caseins, on calcium uptake by human HT-29 intestinal tumor cells, which undergo an enterocytically oriented differentiation in culture. A commercial preparation containing a mixture of purified CPP and an individual CPP of 25 amino acids, both containing the characteristic Ca(2+) binding motif, ser(P)-ser(P)-ser(P)-glu-glu, were employed. The study was performed at the single-cell level and on a cell population and measured the changes in cytosolic calcium concentration before and after CPP addition. In the presence of 2 mmol/L extracellular calcium, both CPP preparations induced a transient rise of free intracellular calcium ions, which did not influence ATP-induced release of calcium from intracellular stores, and which disappeared completely in the absence of extracellular calcium. Pretreatment of these cells with thapsigargin, which completely empties the intracellular calcium stores, did not abolish the cell responses to CPP. Repetitive stimulation of HT-29 cells with CPP always elicited a transient calcium rise, suggesting a lack of desensitization. The CPP-stimulated cytosolic calcium rise was dependent on CPP dose, in a seemingly nonsaturating mode, and on cell numbers. All of this is consistent with the hypothesis that CPP do not influence membrane-bound receptors or ion channels, but may act as calcium ionophores or calcium carriers across the membrane. The reported findings provide a new basis on which to assess the possibility that CPP enhance calcium absorption and bioavailability in animals.

Calcium↗

PubMed2000

Identification and expression of NEU3, a novel human sialidase associated to the plasma membrane.

Several mammalian sialidases have been described so far, suggesting the existence of numerous polypeptides with different tissue distributions, subcellular localizations and substrate specificities. Among these enzymes, plasma-membrane-associated sialidase(s) have a pivotal role in modulating the ganglioside content of the lipid bilayer, suggesting their involvement in the complex mechanisms governing cell-surface biological functions. Here we describe the identification and expression of a human plasma-membrane-associated sialidase, NEU3, isolated starting from an expressed sequence tag (EST) clone. The cDNA for this sialidase encodes a 428-residue protein containing a putative transmembrane helix, a YRIP (single-letter amino acid codes) motif and three Asp boxes characteristic of sialidases. The polypeptide shows high sequence identity (78%) with the membrane-associated sialidase recently purified and cloned from Bos taurus. Northern blot analysis showed a wide pattern of expression of the gene, in both adult and fetal human tissues. Transient expression in COS7 cells permitted the detection of a sialidase activity with high activity towards ganglioside substrates at a pH optimum of 3.8. Immunofluorescence staining of the transfected COS7 cells demonstrated the protein's localization in the plasma membrane.

Alternative Splicing↗

PubMed2000

Membrane anchoring and surface distribution of glycohydrolases of human erythrocyte membranes.

The membrane anchoring of the following glycohydrolases of human erythrocyte plasma membranes was investigated: alpha- and beta-D-glucosidase, alpha- and beta-D-galactosidase, beta-D-glucuronidase, N-acetyl-beta-D-glucosaminidase, alpha-D-mannosidase, and alpha-L-fucosidase. Optimized fluorimetric methods for the assay of these enzymes were set up. Treatment of the ghost preparation with 1.0 mol/l (optimal concentration) NaCl caused release ranging from 4.2% of alpha-D-glucosidase to 70% of beta-D-galactosidase; treatment with 0.4% (optimal concentration) Triton X-100 liberated 5.1% of beta-D-galactosidase to 89% of alpha-D-glucosidase; treatment with 1.75% (optimal concentration) octylglucoside yielded solubilization from 6.3% of beta-D-galactosidase to 85% of alpha-D-glucosidase. Treatment with phosphoinositide-specific phospholipase C caused no liberation of any of the studied glycohydrolases. These results are consistent with the notion that the above glycohydrolases are differently anchored or associated with the erythrocyte plasma membrane, and provide the methodological basis for inspecting the occurrence of these enzymes in different membrane microdomains.

Adult↗

PubMed2000

Sphingolipid-enriched membrane domains from rat cerebellar granule cells differentiated in culture. A compositional study.

Sphingolipid-enriched membrane domains, characterized by a particular protein and lipid composition, have been detected in a variety of cells. However, limited data are available concerning these domains in neuronal cells. We analyzed the lipid and protein composition of a sphingolipid-enriched membrane fraction prepared from primary rat cerebellar granule cells differentiated in culture. Although the protein content of this fraction was only 1.4% of total cellular protein, 60% of the gangliosides, 67% of the sphingomyelin, 50% of the ceramide, and 40% of the cholesterol were located in this fraction. The protein pattern of the sphingolipid-enriched domain fraction was dramatically different from that associated with the cell homogenate. This fraction contained 25% of the tyrosine-phosphorylated proteins and was enriched in two proteins with apparent molecular masses of 135 and 15 kDa. 12% of cellular glycerophospholipids were located in the fraction, with phosphatidylcholine having the highest enrichment. The molar ratio between proteins, glycerophospholipids, cholesterol, sphingomyelin, ceramide and gangliosides in cerebellar granule cells was 1.6:41.6:6. 1:1.3:0.3:1 in the cell homogenate and 0.04:8.3:4.0:1.4:0.2:1 in the sphingolipid-enriched membrane fraction. These data indicate that selected proteins segregate with sphingolipids in specialized domains in the membrane of cultured neurons.

Animals↗