A micro-PET/CT approach using O-(2-[18F]fluoroethyl)-L-tyrosine in an experimental animal model of F98 glioma for BNCT.

The present study focuses on a micro-PET/CT application to be used for experimental Boron Neutron Capture Therapy (BNCT), which integrates, in the same frame, micro-CT derived anatomy and PET radiotracer distribution. Preliminary results have demonstrated that (18)F-fluoroethyl-tyrosine (FET)/PET allows the identification of the extent of cerebral lesions in F98 tumor bearing rat. Neutron autoradiography and α-spectrometry on axial tissues slices confirmed the tumor localization and extraction, after the administration of fructose-boronophenylalanine (BPA). Therefore, FET-PET approach can be used to assess the transport, the net influx, and the accumulation of FET, as an aromatic amino acid analog of BPA, in experimental animal model. Coregistered micro-CT images allowed the accurate morphological localization of the radiotracer distribution and its potential use for experimental BNCT.

Computer sequence analysis of human highly conserved zinc finger modules.

We defined a sub-family of zinc finger proteins by computer analyses and comparisons of five new finger domains against protein databases. This subclass of the cysteine-cysteine/histidine-histidine motif shows additional well conserved amino acid patterns and belongs to the human kox and gli-Kruppel gene family, sharing also the same stretches of regulatory zinc finger-containing proteins of mouse and Xenopus. We particularly describe ZF6 cDNA which contains the most interesting sequence, encoding a putative multi-domain regulatory protein.

Minimal residual disease in B-lymphoproliferative disorders by PCR detection of immunoglobulin heavy chain gene recombination.

We amplified and sequenced the rearranged immunoglobulin heavy chain VDJ genomic unit in B-leukemias and used it as a clone-specific marker for the molecular monitoring of the patients during and after therapeutic treatment. The method described is patient-specific rather than disorder-specific, more sensitive and less time-consuming than other conventional techniques for the detection of minimal residual disease. We propose reproducible and quick procedures, from DNA extraction to Southern blotting, that can be easily performed in any clinical laboratory and also applied to other kinds of investigation.

Different hematological phenotypes caused by the interaction of triplicated alpha-globin genes and heterozygous beta-thalassemia.

The pathophysiology and clinical severity of beta-thalassemia are related to the degree of alpha/non-alpha-chain imbalance. A triplicated alpha-globin gene locus can exacerbate effects of excess alpha-chains caused by a defective beta-globin gene, although this is not observed in all cases. Extensive studies on this condition are lacking. We report a group of 17 patients who are heterozygous for both the alpha alpha alpha(anti-3.7) allele and a mutation in the beta-globin gene cluster. Their clinical phenotypes varied: six had mild anemia with microcytosis and hypochromia, while 11 had more severe anemia with splenomegaly requiring splenectomy (three cases) and blood transfusions (four cases). Different phenotypes were also evident in the presence of the same beta-thalassemia mutation: in one family, two individuals had the same alpha- and beta-globin genotypes but presented with different hematologic phenotypes. In addition, the complex interaction involving a triplicated alpha-globin gene, beta39- and delta+27-thalassemia mutations is studied in a family with two siblings presenting with hemolytic anemia, normal Hb A2 and increased Hb F. Analysis of this series of patients suggests that additional genetic determinants play a role in modulating phenotypic expression in individuals with identical alpha- and beta-globin genotypes. Interaction with a triplicated alpha-gene can play a role in the clinical presentation of patients with defective beta-globin gene expression and should be considered in the diagnosis of atypical cases.

Use of the Oximeter-539 WTW, equipped with a sensor Trioxmatic-300, for the functional analysis of dilute solutions of human Hb-A.

Dissociation curves for oxygen of dilute samples of human adult Hb-A were evaluated on this occasion, by using the Oximeter-539 WTW with its sensor, and a suitable spectrophotometer. At this purpose, Hb samples were desaturated in oxygen upon given experimental conditions, by bubbling pure nitrogen in them, and their re-oxigenation in air was followed, step by step, by multiple oximetries. In addition, all the spectrophotometric measurements of the saturation of Hb-O2%, corresponding to each individual oximetry, were carried out parallely but separately. Dilution of Hb-A was maintained at 0.1 mM in heme. The p50 at pH 7.3 was 4.435 +/- 0.299 Torr, with the n-value of 2.7 +/- 0.2; Bohr effect was -0.55 +/- 0.08, within a pH range between 6.8, 7.3 and 7.8, whereas chloride and DPG effects at pH 7.3 (the most useful value) were 0.42 +/- 0.44 and 0.453 +/- 0.0187 respectively. In conclusion, these results are similar to those obtained with automated procedures, upon comparable experimental conditions, but do not require expensive and sophisticated instruments. Such a technique could be very useful in the hemoglobinopathies, which are common in Italy, and it could be easily adapted to perform comparative studies on animal hemoglobins not far from human species.

P1GF-saporin fusion protein: a potential anti-angiogenic agent.

Vascularization is an important step in tumor growth and metastasis. Tumor neovascularization can be considered, therefore, as a good target for antineoplastic therapy. In order to target saporin, a powerful plant toxin, in proximity of the tumor we fused the saporin coding sequence to that for placental growth factor-2 (P1GF-2). P1GF is an angiogenic factor involved in tumor neovascularization. The fusion protein P1GF-2-saporin was obtained by transient transfection of mammalian cells and released in the culture medium as a 57.5 kDa polypeptide. Selectivity and cytotoxic activity are reported as a preliminary step towards the evaluation of its in vivo antitumor activity.

Mutations in the P53 and RAS family genes are associated with tumor progression of BCR/ABL negative chronic myeloproliferative disorders.

We have investigated the involvement of the p53 tumor suppressor gene and RAS family proto-oncogenes in BCR/ABL-negative chronic myeloproliferative disorders (CMPD), including nine cases of myelosclerosis with myeloid metaplasia, four polycythemia vera, 10 essential thrombocythemia, one juvenile chronic myeloid leukemia, and eight BCR/ABL-negative chronic myeloid leukemia. Twenty-five samples were studied in the chronic phase, while seven samples were analyzed in the acute accelerated or blastic phase. The presence of mutations in p53 exons 5-9, as well as in N-, K-, H-Ras exons 1 and 2 (containing codons 12, 13, and 61) was tested by the polymerase chain reaction (PCR) single strand conformation polymorphism technique and by PCR direct sequencing. In addition, restriction analysis was performed to screen for gross rearrangements within the p53 locus. Alterations of the p53 tumor suppressor gene and Ras family proto-oncogenes were detected in 2/7 and 3/7 cases of acute phase BCR/ABL-negative CMPD, respectively, while consistently negative in all the chronic phase samples analyzed. These results suggest that p53 inactivation and/or Ras activation might play a role in acute transformation of BCR/ABL-negative CMPD.

Molecular defects associated with the acute phase CML.

Parts of the Bcr/Abl hybrid transcript supposed to be important for its transforming ability were sequenced in a series of CML blast crises, in order to evaluate the possible presence of alterations responsible for the disease transition from the chronic to the acute phase. In addition, the N- and Ki-ras as well as the p53 involvement was investigated by exploring their structure and expression in the same patients. We used traditional types of molecular analysis including Southern and Northern blot, together with methods that allow a rapid detection of point mutations and microdeletions, such as SSCP, single strand conformation polymorphism and direct sequencing. The results obtained may be summarized as follows: no alterations were found in the parts of the Bcr/Abl transcripts investigated in the present study (SH2, SH3 and the region surrounding codon 832); p53 alterations were observed in 5% and N- and Ki-RAS mutations in 5% of the cases examined. These molecular defects are therefore responsible for the clinical progression of the Ph1-positive CML only in a minority of cases.

Tumorigenic activity of a rearranged c-myc gene from a human T-cell leukemia line.

The T-lymphoma cell line Hut78 contains a rearranged c-myc oncogene derived from a translocation between the long arms of chromosomes 8 and 2; the event deletes the 3' end of the gene, causing the loss of the transcribed AT-rich sequence. It has recently been shown that the mutant c-myc mRNA is several-fold more stable than normal c-myc mRNA. We have assessed the tumorigenicity of the mutant c-myc allele by transfecting this gene and its normal counterpart into NIH3T3 cells, together with a neomycin resistance gene. Following selection for G-418 resistance, the cells were injected into nude mice. Tumors containing integrated c-myc arose in animals injected with cells transfected by the mutated, but not by the normal, allele. The results suggest that this rearranged c-myc bears a tumorigenic activity not observed in other naturally occurring mutated c-myc alleles and may have directly contributed to the tumorigenic event in the Hut78 cell line.

Characterization of a subfamily of zinc finger genes expressed in human hematopoietic cell lines.

We isolated by low stringency screening of a human erythroleukemia cDNA library (K562) 45 independent clones hybridizing to a Krüppel-like (HF.10) zinc finger cDNA. The expression of 15 such cDNAs in human hematopoietic cell lines was investigated. Preliminary sequence analysis of the zinc finger motifs in these cDNAs indicate that they belong to a subclass of the Cys-Cys/His-His motif, showing the highest homology to the Wilm's tumor and EGR1, EGR2 cDNAs.

Characterization of a subfamily of zinc finger genes expressed in human hematopoietic cell lines.

We isolated by low strigency screening of a human erythroleukemia cDNA library (K562) 45 indipendent clones hybridizing to a Kruppel-like (HF.10) zinc finger cDNA. The expression of 15 such cDNAs in human hematopoietic cell lines was investigated. Preliminary sequence analysis of the zinc finger motifs in these cDNAs indicate that they belong to a subclass of the Cys-Cys/His-His motif, showing the highest homology to the Wilm's tumor and EGR1, EGR2 cDNAs.