Changes in gene expression of DOR and other thyroid hormone receptors in rat liver during acute-phase response

Non-thyroidal illness is characterized by low tri-iodothyronine (T3) serum level under acute-phase conditions. We studied hepatic gene expression of the newly identified thyroid hormone receptor (TR) cofactor DOR/TP53INP2 together with TRs in a rat model of aseptic abscesses induced by injecting intramuscular turpentine-oil into each hind limb. A fast (4-6 h) decrease in the serum level of free thyroxine and free T3 was observed. By immunohistology, abundant DOR protein expression was detected in the nuclei of hepatocytes and ED-1+ (mononuclear phagocytes), CK-19+ (biliary cells), and SMA+ (mesenchymal cells of the portal tract) cells. DOR signal was reduced with a minimum at 6-12 h after the acute-phase reaction (APR). Immunohistology also showed a similar pattern of protein expression in TRα1 but without a significant change during APR. Transcripts specific for DOR, nuclear receptor co-repressor 1 (NCoR-1), and TRβ1 were down-regulated with a minimum at 6-12 h, whereas expression for TRα1 and TRα2 was slightly and significantly up-regulated, respectively, with a maximum at 24 h after APR was initiated. In cultured hepatocytes, acute-phase cytokines interleukin-1β (IL-1β) and IL-6 down-regulated DOR and TRβ1 at the mRNA level. Moreover, gene expression of DOR and TRs (TRα1, TRα2, and TRβ1) was up-regulated in hepatocytes by adding T3 to the culture medium; this up-regulation was almost completely blocked by treating the cells with IL-6. Thus, TRβ1, NCoR-1, and the recently identified DOR/TP53INP2 are abundantly expressed and down-regulated in liver cells during APR. Their down-regulation is attributable to the decreased serum level of thyroid hormones and most probably also to the direct action of the main acute-phase cytokines

Comparison of changes in gene expression of transferrin receptor-1 and other iron-regulatory proteins in rat liver and brain during acute-phase response

The “acute phase” is clinically characterized by homeostatic alterations such as somnolence, adinamia, fever, muscular weakness, and leukocytosis. Dramatic changes in iron metabolism are observed under acute-phase conditions. Rats were administered turpentine oil (TO) intramuscularly to induce a sterile abscess and killed at various time points. Tissue iron content in the liver and brain increased progressively after TO administration. Immunohistology revealed an abundant expression of transferrin receptor-1 (TfR1) in the membrane and cytoplasm of the liver cells, in contrast to almost only nuclear expression of TfR1 in brain tissue. The expression of TfR1 increased at the protein and RNA levels in both organs. Gene expression of hepcidin, ferritin-H, iron-regulatory protein-1, and heme oxygenase-1 was also upregulated, whereas that of hemojuvelin, ferroportin-1, and the hemochromatosis gene was significantly downregulated at the same time points in both the brain and the liver at the RNA level. However, in contrast to observations in the liver, gene expression of the main acute-phase cytokine (interleukin-6) in the brain was significantly upregulated. In vitro experiments revealed TfR1 membranous protein expression in the liver cells, whereas nuclear and cytoplasmic TfR1 protein was detectable in brain cells. During the non-bacterial acute phase, iron content in the liver and brain increased together with the expression of TfR1. The iron metabolism proteins were regulated in a way similar to that observed in the liver, possibly by locally produced acute-phase cytokines. The significance of the presence of TfR1 in the nucleus of the brain cells has to be clarified

Binding and Uptake into Human Hepatocellular Carcinoma Cells of Peptide-Functionalized Gold Nanoparticles

One of the most daunting challenges of nanomedicine is the finding of appropriate targeting agents to deliver suitable payloads precisely to cells affected by malignancies. Even more complex is to achieve the ability to ensure the nanosystems enter those cells. Here we use 2 nm (metal core) gold nanoparticles to target human hepatocellular carcinoma (HepG2) cells stably transfected with the SERPINB3 (SB3) protein. The nanoparticles were coated with a 85:15 mixture of thiols featuring, respectively, a phosphoryl choline, to ensure water solubility and biocompatibility, and a 28-mer peptide corresponding to the amino acid sequence 21-47 of the hepatitis B virus-PreS1 protein (PreS1(21-47)). Conjugation of the peptide was performed via the maleimide-thiol reaction in methanol allowing the use of a limited amount of the targeting molecule. This is an efficient procedure also in the perspective of selecting libraries of new targeting agents. The rationale behind the selection of the peptide is that SB3, which is undetectable in normal hepatocytes, is over-expressed in hepatocellular carcinoma and in hepatoblastoma and has been proposed as a target of the hepatitis B virus (HBV). For the latter the key recognition element is the PreS1(21-47) peptide, which is a fragment of one of the proteins composing the viral envelope. The ability of the conjugated nanoparticles to bind the target protein SB3, expressed in liver cancer cells, was investigated by surface plasmon resonance analysis and in vitro via cellular uptake analysis followed by atomic absorption analysis of digested samples. The results showed that the PreS1(21-47) peptide is a suitable targeting agent for cells overexpressing the SB3 protein. Even more important is the evidence that the gold nanoparticles are internalized by the cells. The comparison between the surface plasmon resonance analysis and the cellular uptake studies suggests the presentation of the protein on cell surface is critical for efficient recognition

Altered regulation of Prox1-gene-expression in liver tumors

This is an Open Access article distributed under the terms of the Creative Commons Attribution Licens

Synthesis and elaboration of peptidomimetic units

Retro-peptides are peptidomimetics that present the direction of the amino acidic sequence reversed. A classical retro modification can be obtained by the introduction of the malonyl unit followed by a gem-diaminic unit, that can result in an increase of proteolysis resistance of the related parent peptide, retarding degradation and consequently enhancing therapeutic efficacy.This thesis aims at developing synthetic stategies to synthesize new interesting building blocks for the construction of peptidomimetics containing a retro modification.In this field Meldrum’s acid is recently reported as a useful scaffold to obtain non-symmetric disubstituted malonamides rAA-mGly-AA′ as building blocks for the synthesis of retro-peptides. The attention has been turned towards the synthetic elaboration of different rAA-mGly-AA′ with the aim of introducing, into the malonamide backbone, the olefinic moiety by a Knoevenagel condensation, obtaining correspondig malonyl dehydro peptides MDHPs.The investigation of a further elaboration was carried out to obtain the corresponding epoxy and aziridino peptides,interesting building blocks containing electrophilic sites known to react with nucleophilic amino acids within the active site of proteases. Starting from L-amino acids, a new methodology is presented for the synthesis of gem-diaminic units g-AA that replace the sense of direction of amino acids in retro-peptide chain.In the field of amino acid and peptide modifications by palladium catalysed reactions, in the University of Bath a methodology has been developed to synthesise biphenyl lysine derivatives by a two step procedure, where the key step involves a Suzuki cross-coupling reaction

Synthesis and elaboration of peptidomimetic units

Retro-peptides are peptidomimetics that present the direction of the amino acidic sequence reversed. A classical retro modification can be obtained by the introduction of the malonyl unit followed by a gem-diaminic unit, that can result in an increase of proteolysis resistance of the related parent peptide, retarding degradation and consequently enhancing therapeutic efficacy.This thesis aims at developing synthetic stategies to synthesize new interesting building blocks for the construction of peptidomimetics containing a retro modification.In this field Meldrum’s acid is recently reported as a useful scaffold to obtain non-symmetric disubstituted malonamides rAA-mGly-AA′ as building blocks for the synthesis of retro-peptides. The attention has been turned towards the synthetic elaboration of different rAA-mGly-AA′ with the aim of introducing, into the malonamide backbone, the olefinic moiety by a Knoevenagel condensation, obtaining correspondig malonyl dehydro peptides MDHPs.The investigation of a further elaboration was carried out to obtain the corresponding epoxy and aziridino peptides,interesting building blocks containing electrophilic sites known to react with nucleophilic amino acids within the active site of proteases. Starting from L-amino acids, a new methodology is presented for the synthesis of gem-diaminic units g-AA that replace the sense of direction of amino acids in retro-peptide chain.In the field of amino acid and peptide modifications by palladium catalysed reactions, in the University of Bath a methodology has been developed to synthesise biphenyl lysine derivatives by a two step procedure, where the key step involves a Suzuki cross-coupling reaction

Modulation of Chemokine- and Adhesion-Molecule Gene Expression and Recruitment of Neutrophil Granulocytes in Rat and Mouse Liver after a Single Gadolinium Chloride or Zymosan Treatment

Kupffer cells are professional phagocytes of the liver clearing bacteria from portal blood. Their clearance capacity, however, can be overwhelmed, transforming them into critical mediators of hepatic-injury. We investigated the consequences of selective Kupffer cell-overload by intraperitoneally administering pyrogen-free gadolinium chloride (GdCl3) or Zymosan into rats and into endotoxin-resistant mice (C3H/HeJ). The number of myeloperoxidase-positive (MPO+) cells increased at 3 h mainly around the portal vessel after both GdCl3 and Zymosan treatment. Simultaneously, GdCl3 administration reduced detectability of ED-1+ (but not ED-2) cells near the portal vessel. Serum chemokine (C-X-C motif) ligand 1 (CXCL-1), CXCL-2 and chemokine (C-C motif) ligand 2 (CCL-2) showed a peak at 3 h after both treatment regimens although at a higher extent after Zymosan administration. Accordingly, CXCL-1, CXCL-5 and CCL-2 gene expression in the liver was up-regulated after GdCl3 treatment at 3 h. After Zymosan administration a significant up-regulation of CXCL-1, CXCL-2, CXCL-10, CCL-2, CCL-3 and CCL-20 gene expression in liver at 3 h was observed. After Zymosan administration intracellular adhesion molecule 1 (ICAM-1) and vascular cell adhesion molecule 1 (VCAM-1) gene expression was up-regulated in rat liver tissue. In C3H/HeJ mice both treatment regimens up-regulated CCL-2 and ICAM-1 gene expression after 3 h and down-regulated platelet endothelial cell adhesion molecule 1 (PECAM-1) gene expression. In conclusion, phagocytosis overload of Kupffer cells causes induction of several CXC, CC-chemokines, upregulation of “positive” adhesion molecule gene expression, down-regulation of the “negative” adhesion molecule PECAM-1 and a recruitment of neutrophil granulocytes in the portal area of the liver of treated rats and mice mainly in close contact to the liver macrophages

\u201cNMR Chemosensing\u201d Using Monolayer-Protected Nanoparticles as Receptors

A new sensing protocol based on NMR magnetization transfer sequences and the molecular recognition abilities of nanoparticles allows the detection and identification of organic molecules in complex mixtures

Metodo di Rilevamento di Molecole Organiche Mediante Spettroscopia di Risonanza Magnetica Nucleare e Nanoparticelle di Oro

L\u2019invenzione \ue8 relativa ad un metodo di rilevamento di molecole organiche mediante spettroscopia NMR a trasferimento di magnetizzazione da nanoparticelle d\u2019oro funzionalizzate con un monostrato di molecole organiche e all\u2019uso delle nanoparticelle d\u2019oro funzionalizzate per il metodo stesso