Expression of the rat homologue of the Drosophila fat tumour suppressor gene.

We have sequenced and defined the expression during rat embryogenesis of the protocadherin fat, the murine homologue of a Drosophila tumour suppressor gene. As previously described for human fat, the sequence encodes a large protocadherin with 34 cadherin repeats, five epidermal growth factor (EGF)-like repeats containing a single laminin A-G domain and a putative transmembrane portion followed by a cytoplasmic sequence. This cytoplasmic sequence shows homology to the b-catenin binding regions of classical cadherin cytoplasmic tails and also ends with a PDZ domain-binding motif. In situ hybridization studies at E15 show that fat is predominately expressed in fetal epithelial cell layers and in the CNS, although expression is also seen in tongue musculature and condensing cartilage. Within the CNS, expression is seen in the germinal regions and in areas of developing cortex, and this neural expression pattern is also seen at later embryonic (E18) and postnatal stages. No labelling was seen in adult tissues except in the CNS, where the remnant of the germinal zones, as well as the dentate gyrus, continue to express fat.

V3 loop core region serotyping of HIV-1 infected patients using the FHV epitope presenting system.

We have reported recently a new epitope presenting system based on the Flock House Virus (FHV) capsid protein. The HIV-1 V3 loop core sequence IGPGRAF was inserted in different sites of this carrier molecule. Immunoreactivity experiments and molecular modelling consistently showed that the most reactive recombinant protein displayed the IGPGRAF sequence in a conformation which is most similar to that of a V3 loop reference structure. The same insertion site was then used to display the V3 loop apex sequences of six different HIV-1 isolates. Sera from 32 HIV-1 infected patients were examined for their reactivity to our chimeric proteins and the results were compared with those obtained using synthetic V3 loop peptides. The data obtained were confirmed by nested PCR amplification and direct sequencing of the patient's V3 loops. The results showed that the V3 loop serotyping using the FHV hybrid proteins, was more specific than that obtained using synthetic peptides. This system will therefore be a useful tool for the correct evaluation of the immune response against different V3 loop core sequences.

Carborane derivatives loaded into liposomes as efficient delivery systems for boron neutron capture therapy.

Boron neutron capture therapy (BNCT) is an anticancer therapy based on the incorporation of (10)B in tumors, followed by neutron irradiation. Recently, the synthesis and delivery of new boronated compounds have been recognized as some of the main challenges in BNCT application. Here, we report on the use of liposomes as carriers for BNCT active compounds. Two carborane derivatives, i.e., o-closocarboranyl beta-lactoside (LCOB) and 1-methyl-o-closocarboranyl-2-hexylthioporphyrazine (H(2)PzCOB), were loaded into liposomes bearing different surface charges. The efficacy of these formulations was tested on model cell cultures, that is, DHD/K12/TRb rat colon carcinoma and B16-F10 murine melanoma. These induce liver and lung metastases, respectively, and are used to study the uptake of standard BNCT drugs, including borophenylalanine (BPA). Boron concentration in treated cells was measured by alpha spectrometry at the TRIGA mark II reactor (University of Pavia). Results showed high performance of the proposed formulations. In particular, the use of cationic liposomes increased the cellular concentration of (10)B by at least 30 times more than that achieved by BPA.

High-mobility-group (HMG) proteins and histone H1 subtypes expression in normal and tumor tissues of mouse.

Exhaustive extraction of mouse tissues with perchloric acid has been used together with reverse-phase HPLC and electrophoresis to quantify the amounts of chromosomal proteins HMG17, HMG14 and HMGI, relative to histone H1. Normal lung and thymus contain approximately 3% HMG17/HMG14 but only approximately 2% HMGI. In tumor tissues (Lewis lung carcinoma and lymphoma NQ35), the amount of HMG17/HMG14 is not greatly altered but HMGI levels rise considerably, reaching 10% in Lewis lung carcinoma. HMGI synthesis does not replace HMG17/HMG14 proteins, suggesting that HMGI proteins contribute to the structure of chromatin regions in a manner distinct from those of HMG17/HMG14. Ion-spray mass spectrometry has been used to determine the molecular masses of H1 subtypes from the same four mouse tissues. In addition to the six known species H1 zero, H1a, H1b, H1c, H1d and H1e, a newly defined subtype of mass 21,756 Da from Lewis lung carcinoma, named H1L was identified. Several phosphorylated H1 subtypes have also been defined by mass spectrometry. The combined use of reverse-phase HPLC and electrophoresis permitted quantification of these seven histone H1 subtypes in the four mouse tissues. Increased phosphorylation of H1 subtypes in tumors parallels the phosphorylation of HMGI proteins which are present in great amounts, showing that both are involved as post-translational-modified forms in the structure of the chromatin of neoplastic systems.