Effects of a hydroxyapatite-based biomaterial on gene expression in osteoblast-like cells.

Biostite is a hydroxyapatite-derived biomaterial that is used in periodontal and bone reconstructive procedures due to its osteoconductive properties. Since the molecular effects of this biomaterial on osteoblasts are still unknown, we decided to assess whether it may specifically modulate osteoblast functions in vitro. We found that a brief exposure to Biostite significantly reduced the proliferation of MG-63 and SaOS-2 osteoblast-like cells to approximately 50% of the plateau value. Furthermore, gene array analysis of MG-63 cells showed that Biostite caused a differential expression of 37 genes which are involved in cell proliferation and interaction, and related to osteoblast differentiation and tissue regeneration. Results were confirmed by RT-PCR, Western blot, and by an increase in alkaline phosphatase (ALP) specific activity. Biostite also increased levels of polycystin-2, a mechano-sensitive Ca(2+) channel, a promising new marker of bone cell differentiation. Biostite, therefore, may directly affect osteoblasts by enhancing chondro/osteogenic gene expression and cytoskeleton-related signaling pathways, which may contribute to its clinical efficacy.

Preferential expression of the transcription coactivator HTIF1alpha gene in acute myeloid leukemia and MDS-related AML.

HTIF1alpha, a transcription coactivator which is able to mediate RARalpha activity and functionally interact with PML, is encoded by a gene on chromosome 7q32-34, which is a critical region in acute myeloid leukemias (AML). With the assumption that this gene may be related to AML, we investigated the HTIF1alpha DNA structure and RNA expression in leukemic cells from 36 M1-M5 AML patients (28 "de novo" and eight "secondary" to myelodysplastic syndrome (MDS)). Abnormal HTIF1alpha DNA fragments were never found, whereas loss of HTIF1alpha DNA was observed in the patients with chromosome 7q32 deletion and translocation, and in one case without detectable chromosome 7 abnormality. HTIF1alpha RNA was found in acute myelocytic leukemic blasts, and was almost undetectable in normal mononuclear cells. The expression varied among the patients: higher in M1 to M3 subtypes, with the highest values in M1; low levels were constantly observed in M4 and M5 AML. In addition, HTIF1alpha was significantly overexpressed in MDS-related AML (MDR-AML), but not in MDS. We also found that HTIF1alpha expression was high in myeloid cell lines. In myeloblastic HL60 and promyelocytic NB4 cells, induced to differentiate along the monocytic-macrophage pathway by TPA or vitamin D3, HTIF1alpha expression decreased, whereas it was maintained at high levels on induction to granulocytic differentiation by RA or DMSO. In K562 cells, HTIF1alpha RNA levels did not change after hemin-induced erythroid differentiation. These results suggest that HTIF1alpha could play a role in myeloid differentiation, being distinctly regulated in hematopoietic lineages.

MECP2 gene mutation analysis in the British and Italian Rett Syndrome patients: hot spot map of the most recurrent mutations and bioinformatic analysis of a new MECP2 conserved region.

Rett syndrome (RTT) is an X-linked dominant neurological disorder, which appears to be the most common genetic cause of profound combined intellectual and physical disability in Caucasian females. This syndrome has been associated with mutations of the MECP2 gene, a transcriptional repressor of unknown target genes. We report a detailed mutational analysis of a large cohort of RTT patients from the UK and Italy. This study has permitted us to produce a hot spot map of the mutations identified. Bioinformatic analysis of the mutations, taking advantage of structural and evolutionary data, leads us to postulate the existence of a new functional domain in the MeCP2 protein, conserved among brain-specific regulatory factors.

Mutation analysis of the MECP2 gene in British and Italian Rett syndrome females.

Rett syndrome is an X-linked dominant neurological disorder, which appears to be the commonest genetic cause of profound combined intellectual and physical disability in Caucasian females. Recently, this syndrome has been associated with mutations of the MECP2 gene, a transcriptional repressor of still unknown target genes. Here we report a detailed mutational analysis of 62 patients from UK and Italian archives, representing the first comparative study among different populations and one of the largest number of cases so far analyzed. We review the literature on MECP2 mutations in Rett syndrome. This analysis has permitted us to produce a map of the recurrent mutations identified in this and previous studies. Bioinformatic analysis of the mutations, taking advantage of structural and evolutionary data, leads us to postulate the existence of a new functional domain in the MeCP2 protein, which is conserved among brain-specific regulatory factors.

Mutation and transcription analysis of transthyretin gene in Italian families with hereditary amyloidosis: a putative novel hot spot' in codon 47.

Transthyretin gene mutations are associated with autosomal dominant familial amyloidosis. The commonest phenotype in the patients is peripheral neuropathy, but restrictive cardiomyopathy is also a frequent sign. More than 70 different mutations in the gene have been described. Although these mutations are randomly distributed, some hot spots have also been reported notably at position 6, 30, 33, 58, 109, 119 and 122. A few of these codons contain a CpG dinucleotide. We describe an additional 'hot spot' occurring at codon 47, in which we report one novel and two previously described mutations. This codon, however, does not contain a CpG dinucleotide, suggesting that other mechanisms might be responsible for the allelic heterogeneity. All the reported mutations in codon 47 are located in the exon 2 consensus sequence and are potentially involved in splicing. We performed transcription analysis on two livers obtained from transplanted patients carrying the Ala47 mutation. These livers showed a normally spliced message, indicating that this mutation does not affect splicing.

Mutations in autosomal dominant polycystic kidney disease 2 gene: Reduced expression of PKD2 protein in lymphoblastoid cells.

The polycystic kidney disease 2 (PKD2) gene, encoding a 968-amino acid integral membrane protein with six predicted membrane-spanning domains and intracellular NH2 and COOH termini, is mutated in approximately 15% of the cases of autosomal dominant polycystic kidney disease (ADPKD), a common genetic disease frequently resulting in renal failure. For a better understanding of the cause of this disorder, we searched for mutations in the PKD2 gene in two PKD2-linked families characterized by different clinical phenotypes. A common polymorphism, a nonsense mutation, and a frameshift mutation were found. Both mutations are predicted to produce truncated proteins of 314 and 386 amino acids, arrested at the first extracellular loop of the protein. Restriction enzyme analysis of polymerase chain reaction (PCR) and reverse transcriptase (RT)-PCR products, respectively, showed that mutations cosegregated with the disease and mutated alleles were expressed at the messenger RNA level in lymphoblastoid cell lines. However, in these cells, Western blot analysis showed only PKD2 normal protein, and it was expressed at a lower level than that found in cells without the PKD2 mutation. These findings suggest that in lymphoblastoid cells, the truncated protein product of the mutant allele may not be stable.

K562 erythroid and HL60 macrophage differentiation downregulates polycystin, a large membrane-associated protein.

Polycystin, the PKD1 gene product mutated in autosomal dominant polycystic kidney disease, is a large membrane protein which is important in the differentiation of epithelial tubular structure. Furthermore, PKD1 mRNA is expressed in various tissues and in neoplastic cell lines particularly, suggesting that polycystin might be involved in differentiation and/or proliferation of other cell types. Therefore, in order to investigate such a possible role, polyclonal antibodies against a recombinant polycystin peptide were raised and used to study polycystin expression in human leukemia cell lines committed to differentiation. Using Western blot and laser scanning confocal microscopy analyses, we demonstrated expression of polycystin in erythroleukemia K562 cells as a membrane-associated polypeptide of approximately 450 kDa, mainly localized in cell-cell contacts. Protein size and subcellular distribution were similar to those found in the kidney epithelial KJ29 cell line. In addition, K562 cell erythroid differentiation induced by hemin was characterized by a reduction in polycystin expression, as measured by Western blot and Northern blot analyses. Cytofluorimetric analysis indicated that upon hemin treatment there was a progressive reduction in the number of polycystin-expressing cells as well as in proliferation rate. Furthermore, reduction in proliferating and polycystin-expressing cells was also observed in K562 cells after serum starvation. When serum was added to the serum-deprived cells an increase in cell number as well as in number of polycystin-positive cells was observed. In addition, polycystin, also expressed in promyelocytic leukemia HL60 cells, was downregulated when macrophage differentiation in HL60 was induced by TPA. Therefore, in these leukemic cells downregulation of polycystin appeared to be closely related to reduction in cell proliferation and to induction of differentiation. This suggests that polycystin may play a relevant role in these cell processes.