Screening a cDNA library from human skeletal muscle and cardiac muscle with a cDNA probe derived from junctin led to the isolation of two groups of cDNA clones. The first group displayed a deduced amino acid sequence that is 84% identical to that of dog heart junctin, whereas the second group had a single open reading frame that encoded a polypeptide with a predicted mass of 33 kDa, whose first 78 NH 2 -terminal residues are identical to junctin whereas its COOH terminus domain is identical to aspartyl ␤-hydroxylase, a member of the ␣-ketoglutarate-dependent dioxygenase family. We named the latter amino acid sequence junctate. Northern blot analysis indicates that junctate is expressed in a variety of human tissues including heart, pancreas, brain, lung, liver, kidney, and skeletal muscle. Fluorescence in situ hybridization analysis revealed that the genetic loci of junctin and junctate map to the same cytogenetic band on human chromosome 8. Analysis of intron/exon boundaries of the genomic BAC clones demonstrate that junctin, junctate, and aspartyl ␤-hydroxylase result from alternative splicing of the same gene.The predicted lumenal portion of junctate is enriched in negatively charged residues and is able to bind calcium. Scatchard analysis of equilibrium 45

Circulating miRNAs deserve further investigation in order to explore their potential role in the pathogenesis of preeclampsia. In particular, miR-1233 might represent a potential marker of early sPE.

Summary.The human leukaemic K562 cell line can be induced in vitro to undergo erythroid differentiation by a variety of chemical compounds, including haemin, butyric acid, 5-azacytidine and cytosine arabinoside. Differentiation of K562 cells is associated with an increased expression of embryo-fetal globin genes, such as the z, e and g globin genes. Therefore the K562 cell line has been proposed as a useful in vitro model system to determine the therapeutic potential of new differentiating compounds as well as to study the molecular mechanism(s) regulating changes in the expression of embryonic and fetal human globin genes. Inducers of erythroid differentiation which stimulate gglobin synthesis could be considered for possible use in the experimental therapy of those haematological diseases associated with a failure in the expression of adult b-globin genes. In this paper we demonstrated that the G þ C selective DNA-binding drugs chromomycin and mithramycin were powerful inducers of erythroid differentiation of K562 cells. Erythroid differentiation was associated with an increase in the accumulation of (a) Hb Gower 1 and Hb Portland and (b) g-globin mRNA.

We determined whether peptide nucleic acids (PNAs) are able to interact with NF-B p52 transcription factor. The binding of NF-B p52 to DNA-DNA, DNA-PNA, PNA-DNA, and PNA-PNA hybrid molecules carrying the NF-B binding sites of human immunodeficiency type 1 long terminal repeat was studied by (i) biospecific interaction analysis (BIA) using surface plasmon resonance technology, (ii) electrophoretic mobility shift, (iii) DNase I footprinting, and (iv) UV cross-linking assays. Our results demonstrate that NF-B p52 does not efficiently bind to PNA-PNA hybrids. However, a DNA-PNA hybrid molecule was found to be recognized by NF-B p52, although the molecular complexes generated exhibited low stability. From the theoretical point of view, our results suggest that binding of NF-B p52 protein to target DNA motifs is mainly due to contacts with bases; interactions with the DNA backbone are, however, important for stabilization of the protein-DNA complex. From the practical point of view, our results suggest that DNA-PNA hybrid can be recognized by NF-B p52 protein, although with an efficiency lower than DNA-DNA NF-B target molecules; therefore, our results should encourage studies on modified PNAs in order to develop potential agents for the decoy approach in gene therapy.It is well established that both constitutive and tissue-specific regulation of gene expression is operated at the transcriptional level by the interaction between nuclear proteins (transcription factors) and promoter regions containing DNA elements (transcription signals) that exhibit specific nucleotide sequences (1-4). Several reviews reporting the nucleotide sequences of transcription signals and the relative binding proteins have been published (5-7). The requirement of protein-

Biospecific interaction analysis (BIA) was performed using surface plasmon resonance (SPR) and biosensor technologies to detect genetically modified Roundup Ready soybean gene sequences. We first immobilized, on SA sensor chips, single-stranded biotinylated oligonucleotides containing soybean lectin and Roundup Ready gene sequences, and the efficiency of hybridization to oligonucleotide probes differing in length was determined. Second, we immobilized biotinylated PCR products from nontransgenic soybeans (genomes carrying only the lectin gene), as well as from genetically modified Roundup Ready soybean, and we injected the oligonucleotide probes. Furthermore, we used the sensor chips carrying either lectin and Roundup Ready soybean PCR products or 21-mer oligonucleotide as probes, and we injected both nonpurified and purified asymmetric PCR products. The results obtained show that 13 and 15 mer oligonucleotides are suitable probes to detect genetically modified Roundup Ready soybean gene sequences (either target oligonucleotides or PCR products) under standard BIA experimental conditions. By contrast, when 11 mer DNA probes were employed, no efficient hybridization was obtained. All the SPR-based formats were found to be useful for detection of Roundup Ready gene sequences, suggesting that these procedures are useful for the real-time monitoring of hybridization between target single-stranded PCR products, obtained by using as substrates DNA isolated from normal or transgenic soybeans, and oligonucleotide or PCR-generated probes, therefore enabling a one-step, nonradioactive protocol to perform detection.

Tissue transglutaminase (transglutaminase type 2; TG2) is the most ubiquitously expressed member of the transglutaminase family (EC 2.3.2.13) that catalyzes specific post-translational modifications of proteins through a calcium-dependent acyl-transfer reaction (transamidation). In addition, this enzyme displays multiple additional enzymatic activities, such as guanine nucleotide binding and hydrolysis, protein kinase, disulfide isomerase activities, and is involved in cell adhesion. Transglutaminase 2 has been reported as one of key enzymes that is involved in all stages of carcinogenesis; the molecular mechanisms of action and physiopathological effects depend on its expression or activities, cellular localization, and specific cancer model. Since it has been reported as both a potential tumor suppressor and a tumor-promoting factor, the role of this enzyme in cancer is still controversial. Indeed, TG2 overexpression has been frequently associated with cancer stem cells’ survival, inflammation, metastatic spread, and drug resistance. On the other hand, the use of inducers of TG2 transamidating activity seems to inhibit tumor cell plasticity and invasion. This review covers the extensive and rapidly growing field of the role of TG2 in cancer stem cells survival and epithelial–mesenchymal transition, apoptosis and differentiation, and formation of aggressive metastatic phenotypes.

In several types of thalassemia (including β039-thalassemia), stop codon mutations lead to premature translation termination and to mRNA destabilization through nonsense-mediated decay. Drugs (for instance aminoglycosides) can be designed to suppress premature termination, inducing a ribosomal readthrough. These findings have introduced new hopes for the development of a pharmacologic approach to the cure of this disease. However, the effects of aminoglycosides on globin mRNA carrying β-thalassemia stop mutations have not yet been investigated. In this study, we have used a lentiviral construct containing the β039- thalassemia globin gene under control of the β-globin promoter and a LCR cassette. We demonstrated by fluorescence-activated cell sorting (FACS) analysis the production of β-globin by K562 cell clones expressing the β039-thalassemia globin gene and treated with G418. More importantly, after FACS and high-performance liquid chromatography (HPLC) analyses, erythroid precursor cells from β039-thalassemia patients were demonstrated to be able to produce β-globin and adult hemoglobin after treatment with G418. This study strongly suggests that ribosomal readthrough should be considered a strategy for developing experimental strategies for the treatment of β0-thalassemia caused by stop codon mutations.

Peptide nucleic acids (PNAs) containing an insert of three chiral monomers based on D-lysine ('chiral box') were synthesized and used as probes in Biospecific Interaction Analysis (BIA) for the recognition of DNA containing the W1282X point mutation of the cystic fibrosis gene. Hybridization experiments carried out in solution showed enhanced mismatch recognition when compared with the analogous achiral PNAs and oligonucleotides. The signal intensity was lower, but the selectivity of the Biacore response was found to be much higher than that observed with achiral PNAs. The newly designed chiral PNA probes were also found to hybridize with a 1:1 mixture of normal (N-W1282X) and mutated (M-W1282X) DNA oligomers immobilized on the biosensor, thus allowing discrimination not only between a normal and a mutated sequence (healthy/homozygous), but also between homo- and heterozygous individuals. These results suggest that 'chiral box' PNAs are potential powerful tools for the analysis of single point mutations of biological/biomedical relevance.