Biomedical subjects

Leonardo Rossi

Publications and source records attributed to Leonardo Rossi.

13 recordsLinked to original sources

PubMed2007

New fluorescent 2-phenylindolglyoxylamide derivatives as probes targeting the peripheral-type benzodiazepine receptor: design, synthesis, and biological evaluation.

Fluorescent ligands for the peripheral-type benzodiazepine receptor (PBR) featuring the 7-nitrobenz-2-oxa-1,3-diazol-4-yl moiety were synthesized, based on N,N-dialkyl-2-phenylindol-3-ylglyoxylamides, a potent, selective class of PBR ligands previously described by us. All the new ligands are moderately to highly potent at the PBR, with a complete selectivity over the central benzodiazepine receptor. Results from fluorescence microscopy showed that these probes specifically labeled the PBR at the mitochondrial level in C6 glioma cells.

Animals↗

PubMed2006

DjPiwi-1, a member of the PAZ-Piwi gene family, defines a subpopulation of planarian stem cells.

Planarian regeneration, based upon totipotent stem cells, the neoblasts, provides a unique opportunity to study in vivo the molecular program that defines a stem cell. In this study, we report the identification of DjPiwi-1, a planarian homologue of Drosophila Piwi. Expression analysis showed that DjPiwi-1 transcripts are preferentially accumulated in small cells distributed along the midline of the dorsal parenchyma. DjPiwi-1 transcripts were not detectable after X-ray irradiation by whole mount in situ hybridization. Real time reverse transcriptase polymerase chain reaction analysis confirmed the significant reduction of DjPiwi-1 expression after X-ray treatment. However, the presence of residual DjPiwi-1 transcription suggests that, although the majority of DjPiwi-1-positive cells can be neoblasts, this gene is also expressed in differentiating/differentiated cells. During regeneration DjPiwi-1-positive cells reorganize along the midline of the stump and no accumulation of hybridization signal was observed either in the blastema area or in the parenchymal region beneath the blastema. DjPiwi-1-positive cells, as well as the DjMCM2-expressing neoblasts located along the midline and those spread all over the parenchyma, showed a lower tolerance to X-ray with respect to the DjMCM2-expressing neoblasts distributed along the lateral lines of the parenchyma. Taken together, these findings suggest the presence of different neoblast subpopulations in planarians.

Amino Acid Sequence↗

PubMed2006

Detection of human MCP-4/CCL13 isoforms by SELDI immunoaffinity capture.

Monocyte Chemoattractant Proteins 4 (MCP-4/CCL13) is a member of a distinct, structurally-related subclass of CC chemokines mainly involved in recruitment of eosinphils to inflammatory sites. Recent evidence demonstrates that serum level of this protein strongly increases following high dose IL-2 immunotherapy. The physiological form of human MCP-4/CCL13 has yet to be purified. Therefore, the primary structure of the biologically relevant (mature) form has not been established. By using SELDI immunoaffinity capture technology we describe two mature isoforms both present in serum before and after high-dose IL-2 immunotherapy.

Journal Article↗

PubMed2006

Inflammatory protein profile during systemic high dose interleukin-2 administration.

Systemic interleukin-2 (IL-2) administration induces an assortment of downstream effects whose biological and therapeutic significance remains unexplored mostly because of the methodological inability to globally address their complexity. Protein array analysis of sera from patients with renal cell carcinoma obtained prior and during high-dose IL-2 therapy had previously revealed extensive alterations in expression of the soluble factors analyzed, whose discovery was limited by the number of capture antibodies selected for protein detection. Here, we expanded the analysis to SELDI-TOF-MS and quantitative protein analysis (nephelometry). All cytokines/chemokines detected by protein arrays were below the SELDI detection limit, while novel IL-2-specific changes in expression of acute-phase reactants and high-density lipoprotein metabolites could be identified. Serum amyloid protein A (SAA) and C-reactive protein expression were consistently up-regulated after four doses of IL-2, while other proteins were down-regulated. These findings were confirmed by SELDI immunoaffinity capture and nephelometry. Immunoaffinity capture revealed different, otherwise undetectable, isoforms of SAA. A linear correlation between peak area by SELDI and protein concentration by nephelometry was observed. Overall distinct yet complementary information was obtained using different platforms, which may better illustrate complex phenomena such as the systemic response to biological response modifiers.

Biomarkers↗

PubMed2005

Peripheral benzodiazepine receptor: characterization in human T-lymphoma Jurkat cells.

Peripheral benzodiazepine receptor (PBR) has been considered a promising drug target for cancer therapy, and several ligands have been developed for this purpose. Human T-lymphoma Jurkat cells have been considered as lacking PBR and are often used as negative control to prove the specificity of PBR ligands effects. It is surprising that we evidenced PBR protein expression in this cell line by means of Western blotting and immunocytochemistry assays using specific anti-PBR antibodies. PBR intracellular localization was evidenced in mitochondria and nuclei, as demonstrated by confocal and electron microscopy. The binding of the [(3)H]4'-chloro derivative of diazepam [(3)H]7-chloro-5-(4-chlorophenyl)-1,3-dihydro-1-methyl-2H-1,4-benzodiazepin-2-one (Ro5-4864) and the isoquinoline carboxamide derivative [(3)H]1-(2-chlorophenyl)-N-methyl-N-(1-methylpropyl)-3 isoquinolinecarboxamide (PK11195) evidenced a single class of binding sites with an unusual affinity constant (K(d)) of 1.77 +/- 0.30 and 2.20 +/- 0.20 microM, respectively. The pharmacological profile of the classic ligands showed that PK11195 was the most potent inhibitor in the radioligand binding assays followed by Ro5-4864 and diazepam, whereas clonazepam, a specific ligand for the central-type receptor, showed a K(i) >1.0 x 10(-4) M. By a combined strategy of reverse transcriptase-polymerase chain reaction and Southern blot experiments, we succeeded in isolating and cloning the full-length Jurkat PBR cDNA, called JuPBR. The JuPBR gene showed two single-nucleotide polymorphisms resulting in the two substitutions, Ala147 --> threonine and His162 --> arginine, of PBR amino acidic sequence. In conclusion, for the first time, we demonstrated PBR expression in Jurkat cells: the protein bound classic PBR ligands with micromolar affinity constants and presented a modified amino acidic sequence consequent to the detection of two gene polymorphisms.

Amino Acid Sequence↗

PubMed2005

DjPum, a homologue of Drosophila Pumilio, is essential to planarian stem cell maintenance.

As stem cells are rare and difficult to study in vivo in adults, the use of classical models of regeneration to address fundamental aspects of the stem cell biology is emerging. Planarian regeneration, which is based upon totipotent stem cells present in the adult--the so-called neoblasts--provides a unique opportunity to study in vivo the molecular program that defines a stem cell. The choice of a stem cell to self-renew or differentiate involves regulatory molecules that also operate as translational repressors, such as members of PUF proteins. In this study, we identified a homologue of the Drosophila PUF gene Pumilio (DjPum) in the planarian Dugesia japonica, with an expression pattern preferentially restricted to neoblasts. Through RNA interference (RNAi), we demonstrate that gene silencing of DjPum dramatically reduces the number of neoblasts, thus supporting the intriguing hypothesis that stem cell maintenance may be an ancestral function of PUF proteins.

Amino Acid Sequence↗

PubMed2005

PIGA (N,N-Di-n-butyl-5-chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide), a new mitochondrial benzodiazepine-receptor ligand, induces apoptosis in C6 glioma cells.

Mitochondrial benzodiazepine-receptor (mBzR) ligands constitute a heterogeneous class of compounds that show a pleiotropic spectrum of effects within the cells, including the modulation of apoptosis. In this paper, a novel synthetic 2-phenylindol-3-ylglyoxylamide derivative, N,N-di-n-butyl-5-chloro-2-(4-chlorophenyl)indol-3-ylglyoxylamide (PIGA), which shows high affinity and selectivity for the mBzR, is demonstrated to induce apoptosis in rat C6 glioma cells. PIGA was able to dissipate mitochondrial transmembrane potential (DeltaPsim) and to cause a significant cytosolic accumulation of cytochrome c. Moreover, typical features of apoptotic cell death, such as caspase-3 activation and DNA fragmentation, were also detected in PIGA-treated cells. Our data expand the knowledge on mBzR ligand-mediated apoptosis and suggest PIGA as a novel proapoptotic compound with therapeutic potential against glial tumours, in which apoptosis resistance has been reported to be involved in carcinogenesis.

Animals↗

PubMed2004

Peripheral benzodiazepine receptor ligands: mitochondrial transmembrane potential depolarization and apoptosis induction in rat C6 glioma cells.

The peripheral benzodiazepine receptor (PBR) is a component of a multiprotein complex, located at the contact site between the inner and outer mitochondrial membranes, which constitutes the mitochondrial permeability transition (MPT)-pore. The opening of the MPT-pore, leading to the transmembrane mitochondrial potential (DeltaPsi(m)) dissipation, is a critical event in the mechanism of apoptosis. In the present work, we investigated the ability of the specific PBR ligands, PK 11195 or Ro5-4864, to affect mitochondrial potential and to induce apoptotic cell death in rat C6 glioma cells. Both specific ligands inhibited cell survival in a dose- and time-dependent manner, as assessed by MTS conversion assay, whereas the non-site selective ligand Diazepam or the low-affinity benzodiazepine Clonazepam showed no significant effects. After cell exposure to PK 11195 or Ro5-4864 we evidenced typical alterations of apoptotic cell death such as DNA fragmentation and chromatin condensation assessed by flow cytometric and transmission electron microscopy (TEM) analysis, respectively. Activation of the "effector" caspase-3 confirmed the ability of specific PBR ligands to induce apoptosis. Moreover, PK 11195 and Ro5-4864 induced a decrease of DeltaPsi(m), as evidenced by JC-1 flow cytometry analysis. Our data demonstrate the pro-apoptotic effects of specific PBR ligands on rat C6 glioma cells.

Animals↗

PubMed2004

Djeyes absent (Djeya) controls prototypic planarian eye regeneration by cooperating with the transcription factor Djsix-1.

A conserved network of nuclear proteins is crucial to eye formation in both vertebrates and invertebrates. The finding that freshwater planarians can regenerate eyes without the contribution of Pax6 suggests that alternative combinations of regulatory elements may control the morphogenesis of the prototypic planarian eye. To further dissect the molecular events controlling eye regeneration in planarians, we investigated the role of eyes absent (Djeya) and six-1 (Djsix-1) genes in Dugesia japonica. These genes are expressed in both regenerating eyes and in differentiated photoreceptors of intact adults. Through RNAi studies, we show that Djsix-1 and Djeya are both critical for the regeneration of normal eyes in planarians and genetically cooperate in vivo to establish correct eye cell differentiation. We further demonstrate that the genetic interaction is mediated by physical interaction between the evolutionarily conserved domains of these two proteins. These data indicate that planarians use cooperatively Djsix-1 and Djeya for the proper specification of photoreceptors, implicating that the mechanism involving their evolutionarily conserved domains can be very ancient. Finally, both Djsix-1 and Djeya double-stranded RNA are substantially more effective at producing no-eye phenotypes in the second round of regeneration. This is probably due to the significant plasticity of the planarian model system, based on the presence of a stable population of totipotent stem cells, which ensure the rapid cell turnover of all differentiated cell types.

Amino Acid Sequence↗

PubMed2003

Expression of DjXnp, a novel member of the SNF2-like ATP-dependent chromatin remodelling genes, in intact and regenerating planarians.

SWI/SNF-related complexes include proteins implicated in the regulation of gene expression by chromatin remodelling. We have identified in planarians, invertebrates well-known for their regenerative capability,the cDNA of a novel gene, DjXnp, which encodes a protein of 1,076 amino acids, containing seven helicase domains similar to those found in the SNF2-like family members. Sequence comparison reveals a significant degree of similarity of DjXNP with mammalian XNP/ATRX proteins. In situ hybridization experiments performed on intact and regenerating planarians demonstrated that DjXnp transcripts were distributed in mesenchymal cells and were especially abundant in nerve cells. During anterior regeneration, DjXnp was detected in the blastemal area where the nervous system is newly forming. This expression pattern reveals extensive similarities with that described for mammalian XNP/ATRX, suggesting that these genes may have a conserved function at the cellular level.

Adenosine Triphosphate↗

PubMed2002

Characterization of DeY1, a novel Y-box gene specifically expressed in differentiating male germ cells of planarians.

Y-box proteins are conserved regulatory factors that play a key role in coordinating gene activity with protein synthesis by influencing both the transcription and translation of specific subsets of genes. We report the identification of a novel Y-box gene, DeY1, whose transcripts are found in the testes of sexual planarians. DeY1 is expressed in spermatogonia, spermatocytes and spermatids, while no expression is detected in spermatozoa. No DeY1 transcripts are found in the blastema during regeneration. The subcellular distribution of DeY1 protein was analyzed by electron microscope immunocytochemistry. Immunolabelling was found in the nucleus of spermatogonia, in both the nucleus and the cytoplasm of spermatocytes, and in the cytoplasm of spermatids.

Amino Acid Sequence↗

PubMed2002

The genetic network of prototypic planarian eye regeneration is Pax6 independent.

We report the presence of two Pax6-related genes, Pax6A and Pax6B, which are highly conserved in two planarian species Dugesia japonica and Girardia tigrina (Platyhelminthes, Tricladida). Pax6A is more similar to other Pax6 proteins than Pax6B, which is the most divergent Pax6 described so far. The planarian Pax6 homologs do not show any clear orthology to the Drosophila duplicated Pax6 genes, eyeless and twin of eyeless, which suggests an independent Pax6 duplication in a triclad or platyhelminth ancestor. Pax6A is expressed in the central nervous system of intact planarians, labeling a subset of cells of both cephalic ganglia and nerve cords, and is activated during cephalic regeneration. Pax6B follows a similar pattern, but shows a lower level of expression. Pax6A and Pax6B transcripts are detected in visual cells only at the ultrastructural level, probably because a limited amount of transcripts is present in these cells. Inactivation of both Pax6A and Pax6B by RNA-mediated gene interference (RNAi) inhibits neither eye regeneration nor eye maintenance, suggesting that the genetic network that controls this process is not triggered by Pax6 in planarians.

Amino Acid Sequence↗

PubMedDate not supplied

Advancing cancer biotherapy with proteomics.

Proteomics is becoming increasingly important for cancer biotherapy. The development of high-throughput platforms now allows the analysis of multiple proteins from small quantities of material. While these techniques are being used to discover new biomarkers, they are particularly important for assessing complex biologic processes such as immunotherapy for cancer. Recent advances in this field are reviewed, as well as the use of proteomics to assess the effectiveness and toxicities of high-dose IL-2 cancer therapy. Proteomics is becoming useful in assessing cancer biotherapies and in unraveling their mechanisms of action. High-throughput proteomic technologies have now advanced to a stage where they have the potential to become effective discovery tools for biomarkers/predictors of disease, disease recurrence, and response to therapy.

Humans↗