Biomedical subjects

Federica Riva

Publications and source records attributed to Federica Riva.

9 recordsLinked to original sources

PubMed2006

Expression of c-kit proto-oncogene in canine mastocytoma: a kinetic study using real-time polymerase chain reaction.

KIT receptor, the c-kit gene product, is thought to play a major role in canine mastocytoma, one of the most common neoplastic diseases in dogs. In the present study, the expression of c-kit proto-oncogene in blood and in tumor biopsies from 41 dogs with histologically confirmed mastocytoma at different grades of cellular differentiation and 5 negative control dogs was investigated using real-time (quantitative) reverse transcription-polymerase chain reaction (RRT-PCR). The animals were followed up for over 1 year after surgery in order to characterize the kinetics of c-kit expression in blood. Transcript mRNAs extracted from blood at different time points after surgery and from tumor tissue surgically removed from each dog were used in a quantitative RRT-PCR assay targeting the extracellular coding region of the c-kit gene. Tissues constitutively expressing c-kit (brain and spleen) were used as positive controls. Levels of expression of c-kit were higher in tumor biopsies than in blood; the blood level decreased in the patients between 1 and 3 months after surgery. No KIT expression was detected in blood from the 5 dogs not affected by mastocytoma (negative controls). The RRT-PCR appears to be a suitable method for sensitive and quantitative detection of c-kit gene expression in canine blood and neoplastic tissues. Although c-kit expression levels measured by RRT-PCR do not correlate with prognosis, they confirm that surgery remains the main treatment to reduce circulating mastocytes and that circulating mast cells can be detected even in benign highly differentiated forms of mastocytoma such as grade I.

Animals↗

PubMed2005

A study of mutations in the c-kit gene of 32 dogs with mastocytoma.

Mutations in the intracellular juxtamembrane domain of the c-kit gene in 32 dogs with different grades of histologically confirmed mastocytoma were studied. Transcript RNAs extracted from neoplastic tissue surgically collected from dogs of different breeds and from a negative control were reverse transcribed into complementary DNA and amplified by polymerase chain reaction. The region corresponding to the c-kit juxtamembrane domain was sequenced and compared with GenBank sequences. Two different types of mutations were identified within exon 11: a previously underscribed single-nucleotide substitution and a 6-bp deletion. The c-kit juxtamembrane domain sequences of all dogs were grouped in 3 clusters. No mutations were detected in tissues constitutively expressing c-kit (cerebellum and spleen), obtained from dogs not affected by mastocytoma (controls). All the substitutions were found in dogs bearing grade I or II mast cell tumors; the deletion was detected in 1 dog with grade II mastocytoma.

Animals↗

PubMed2005

Cell kinetic analysis in artificial skin using immunochemical methods.

Cell kinetic studies provide important information on histogenesis in vivo and in vitro. In this regard, specific antibodies to cell cycle-related antigens have been raised and characterized, thus permitting the study of cell kinetics using immunochemical methods. Recent advances in culture technology permitted the generation of human skin equivalents in vitro. We here provide detailed practical procedures for the study of epidermal cell kinetics in a model of artificial skin using immunohistochemistry and flow cytometry. The combined application of both techniques allows a precise detection of tissue growth sites and a quantitative assessment of cell growth. Moreover, simultaneous analysis of differentiation markers and proliferation antigens may be useful to understand molecular mechanisms that regulate tissue growth and development.

Cell Cycle↗

PubMed2004

Intestinal inflammation in mice deficient in Tir8, an inhibitory member of the IL-1 receptor family.

TIR8, also known as single Ig IL-1-related receptor, is a member of the IL-1 receptor/Toll-like receptor (TLR) superfamily, which acts as an intracellular decoy for components of the signaling pathway. Here we report that Tir8 has a unique pattern of expression, which includes mucosal tissues and dendritic cells (DC). Tir8-deficient DC showed increased cytokine production in response to TLR agonists (lipopolysaccharide, CpG oligodeoxynucleotides). Tir8-deficient mice had normal susceptibility to systemic lipopolysaccharide toxicity and to i.p. or s.c. inflammation. However, Tir8-deficient mice were more susceptible to intestinal inflammation. Thus, TIR8 represents a negative pathway of regulation of the IL-1 receptor/TLR system, expressed in epithelial cells and DC, crucial for tuning inflammation in the gastrointestinal tract.

Animals↗

PubMed2004

Distinct pools of proliferating cell nuclear antigen associated to DNA replication sites interact with the p125 subunit of DNA polymerase delta or DNA ligase I.

Proliferating cell nuclear antigen (PCNA) plays an essential role in DNA replication, repair, and cell cycle control. PCNA is a homotrimeric ring that, when encircling DNA, is not easily extractable. Consequently, the dynamics of protein-protein interactions established by PCNA at DNA replication sites is not well understood. We have used DNase I to release DNA-bound PCNA together with replication proteins including the p125-catalytic subunit of DNA polymerase delta (p125-pol delta), DNA ligase I, cyclin A, and cyclin-dependent kinase 2 (CDK2). Interaction with these proteins was investigated by immunoprecipitation with antibodies binding near the interdomain connector loop or to the C-terminal domain of PCNA, respectively, or with antibodies to p125-pol delta or DNA ligase I. PCNA interaction with p125-pol delta or DNA ligase I was detected only by the latter antibodies, and found to be mutually exclusive. In contrast, antibodies to PCNA co-immunoprecipitated only CDK2. A GST-p21(waf1/cip1) C-terminal peptide displaced p125-pol delta and DNA ligase I, but not CDK2, from PCNA. These results suggest that PCNA trimers bound to DNA during the S phase are organized as distinct pools able to bind selectively different partners. Among them, p125-pol delta and DNA ligase I interact with PCNA in a mutually exclusive manner.

Antibodies↗

PubMed2003

Human proliferating cell nuclear antigen, poly(ADP-ribose) polymerase-1, and p21waf1/cip1. A dynamic exchange of partners.

We addressed the analysis of the physical and functional association of proliferating cell nuclear antigen (PCNA), a protein involved in many DNA transactions, with poly(ADP-ribose) polymerase (PARP-1), an enzyme that plays a crucial role in DNA repair and interacts with many DNA replication/repair factors. We demonstrated that PARP-1 and PCNA co-immunoprecipitated both from the soluble and the DNA-bound fraction isolated from S-phase-synchronized HeLa cells. Immunoprecipitation experiments with purified proteins further confirmed a physical association between PARP-1 and PCNA. To investigate the effect of this association on PARP-1 activity, an assay based on the incorporation of radioactive NAD was performed. Conversely, the effect of PARP-1 on PCNA-dependent DNA synthesis was assessed by a DNA polymerase delta assay. A marked inhibition of both reactions was found. Unexpectedly, PARP-1 activity also decreased in the presence of p21waf1/cip1. By pull-down experiments, we provided the first evidence for an association between PARP-1 and p21, which involves the C-terminal part of p21 protein. This association was further demonstrated to occur also in vivo in MNNG (N-methyl-N'-nitro-N-nitrosoguanidine)-treated human fibroblasts. These observations suggest that PARP-1 and p21 could cooperate in regulating the functions of PCNA during DNA replication/repair.

Cells, Cultured↗

PubMed2002

Non-redundant role of the long pentraxin PTX3 in anti-fungal innate immune response.

Pentraxins are a superfamily of conserved proteins that are characterized by a cyclic multimeric structure. The classical short pentraxins, C-reactive protein (CRP) and serum amyloid P component (SAP), are acute-phase proteins produced in the liver in response to inflammatory mediators. Short pentraxins regulate innate resistance to microbes and the scavenging of cellular debris and extracellular matrix components. In contrast, long pentraxins have an unrelated, long amino-terminal domain coupled to the carboxy-terminal pentraxin domain, and differ, with respect to short pentraxins, in their gene organization, chromosomal localization, cellular source, and in their stimuli-inducing and ligand-recognition ability. To investigate the in vivo function of the long pentraxin PTX3, we generated mice deficient in Ptx3 by homologous recombination. Ptx3-null mice were susceptible to invasive pulmonary aspergillosis. Ptx3 binds selected microbial agents, including conidia of Aspergillus fumigatus, and we found that susceptibility of Ptx3-null mice was associated with defective recognition of conidia by alveolar macrophages and dendritic cells, as well as inappropriate induction of an adaptive type 2 response. Thus, the long pentraxin Ptx3 is a secreted pattern-recognition receptor that has a non-redundant role in resistance to selected microbial agents, in particular to the opportunistic fungal pathogen Aspergillus fumigatus.

Animals↗

PubMedDate not supplied

p21CDKN1A does not interfere with loading of PCNA at DNA replication sites, but inhibits subsequent binding of DNA polymerase delta at the G1/S phase transition.

The ability of the cyclin-dependent kinase (CDK) inhibitor p21CDKN1A to interact with PCNA recruited to DNA replication sites was investigated to elucidate the relevance of this interaction in cell cycle arrest. To this end, expression of p21 protein fused to green fluorescent protein (GFP) was induced in HeLa cells. G1 phase cell cycle arrest induced by p21GFP occurred also at the G1/S transition, as shown by cyclin A immunostaining of GFP-positive cells. Confocal microscopy analysis and co-immunoprecipitation studies showed that p21GFP co-localized and interacted with chromatin-bound PCNA and CDK2. GFP-p21 mutant forms unable to bind to PCNA (p21PCNA-) or CDK (p21CDK-) induced cell cycle arrest, although immunoprecipitation experiments showed these mutants to be unstable. Expression of HA-tagged p21wt or mutant proteins confirmed the ability of both mutants to arrest cell cycle. p21(wt)HA and p21CDK-HA, but not p21PCNA-, co-localized and co-immunoprecipitated with chromatin-bound PCNA. Association of p21 to chromatin-bound PCNA resulted in the loss of interaction with the p125 catalytic subunit of DNA polymerase delta (pol delta). These results suggest that in vivo p21 does not interfere with loading of PCNA at DNA replication sites, but prevents, or displaces subsequent binding of pol delta to PCNA at the G1/S phase transition.

CDC2-CDC28 Kinases↗

PubMedDate not supplied

Unique pattern of expression and inhibition of IL-1 signaling by the IL-1 receptor family member TIR8/SIGIRR.

TIR8, also known as single Ig IL-1R-related molecule (SIGIRR), is a member of the IL-1 receptor family. The present study was designed to investigate the expression and function of TIR8. TIR8 was mainly expressed in mouse and human epithelial tissues such as kidney, lung and gut. Resting and activated T and B lymphocytes and monocytes-macrophages expressed little or no TIR8, with the exception of the mouse GG2EE macrophage line. In the kidney, the organ with highest mRNA levels, TIR8 expression was confined to epithelial cells and, in situ, to tubular epithelium. A variety of signals failed to regulate TIR8 expression, but LPS reduced TIR8 mRNA transcripts. An NF-kB driven reporter system was used to investigate the function of TIR8. TIR8 did not activate NF-kB expression alone or in concert with IL-1R1. In contrast, TIR8 inhibited signaling from the IL-1R complex. Inhibition required the intracellular portion of TIR8 but the extracellular domain was dispensable for blocking activity. Thus, TIR8 is a unique member of the IL-1R family, with a distinct pattern of epithelial expression, including the kidney and mucosae, and an inhibitory function on IL-1 signaling.

Animals↗