Maria Zannotti

Gene expression profile analysis in human T lymphocytes from patients with Down Syndrome.

Down Syndrome (DS) is caused by the presence of three copies of the whole human chromosome 21 (HC21) or of a HC21 restricted region; the phenotype is likely to have originated from the altered expression of genes in the HC21. We apply the cDNA microarray method to the study of gene expression in human T lymphocytes with trisomy 21 in comparison to normal cells.

Enhancing effect of lithium and potassium ions on lectin-induced lymphocyte proliferation in aging and down's syndrome subjects

The effect of different concentrations of LiCl or KCl (0.6-20 meq/liter) on PHA-stimulated lymphocytes from young, old, and Downs syndrome subjects was studied. LiCl showed a dramatic enhancing effect on [3H]thymidine incorporation induced by a suboptimal dose of PHA in old subjects and Downs syndrome patients. An increase of [3H]thymidine incorporation in human lymphocytes stimulated by a suboptimal dose of PHA was also observed with KCl. This effect was higher in old subjects than that observed in young and Downs subjects. LiCl and KCl can modulate and partially restore the derangement in early events of mitogen stimulation which seems to be present in lymphocytes from both old and Downs syndrome subjects.

AGE-RELATED INCREASE OF MITOMYCIN C-INDUCED MICRONUCLEI IN LYMPHOCYTES FROM DOWN'S SYNDROME SUBJECTS

Peripheral blood lymphocytes from 7 patients with Downs syndrome (DS; trisomy 21) and 14 healthy age-matched controls were studied for the induction of micronuclei (MN) by the cytokinesis-block method. The spontaneous incidence of MN in lymphocytes from DS subjects was lower than that of control cultures. When lymphocytes were treated with mitomycin C (MMC) at the beginning of the culture period, an increase in MN formation was found in cells from both DS and control subjects. In DS subjects this increase was much more marked than in control donors. This effect had to be ascribed to cells from older DS subjects (37-55 years old), which showed an MMC-induced MN formation that was markedly and significantly higher than that observed in cells from younger (9-16 years old) DS subjects. These data indicate that age has to be considered a major variable when studies on the genetic instability of DS subjects are performed.

Alterations of the Capping Phenomenon on Lymphocytes from Aged and Down’s Syndrome Subjects

The redistribution with the time of concanavalin A (Con A) receptors at one pole of the cell after addition of FITC-Con A - so-called capping - in the peripheral blood lymphocytes from 13 aged subjects (mean: 84 +/- 1 years old), and of 16 noninstitutionalized patients affected by a syndrome of precocious aging, such as Downs syndrome (mean: 17 +/- 2 years old), was studied and compared with a group of 15 normal young people (mean: 23 +/- 2 years old). An opposite alteration in the percentage of capped cells, i.e. a decrease in aged subjects and an increase in Downs syndrome patients, was observed. A derangement of lymphocyte membrane fluidity appears to be present in both groups even if the underlying biochemical defect may be different. However, a similar alteration of the kinetics of the phenomenon was present either in aged or in Downs syndrome subjects. Both groups did not show any significant increase with time of the percentage of capped cells, suggesting that they were lacking a lymphocyte subpopulation(s) which start capping later.

The murine DSCR1-like (Down Syndrome Candidate Region 1) gene family: conserved synteny with the human orthologous genes

A recently recognized gene family, conserved from yeast to humans, includes Down syndrome candidate region 1 gene (DSCR1), Adapt78 (recognized as the hamster ortholog of the DSCR1 isoform 4), ZAKI-4 (renamed DSCR1-like 1, DSCR1L1) and DSCR1L2 (a novel gene on human chromosome 1), along with yeast and C. elegans single members (Strippoli P., Lenzi L., Petrini M., Carinci P., Zannotti M., 2000. A new gene family including DSCR1 (Down Syndrome Candidate Region 1) and ZAKI-4: characterization from yeast to human and identification of DSCR1-like 2, a novel human member. Genomics 64, 252-263). The proposed family labels were a putative single-strand nucleic acid binding domain similar to the RNA recognition motif, and a unique, highly-conserved serine-proline motif. We have used a bioinformatics-driven molecular biology approach to characterize the murine members of DSCR1-like gene family. Systematic expressed-sequence-tags (EST) database search and reverse-transcription polymerase chain rection (RT-PCR) product sequencing allowed identification of the murine DSCR1, DSCR1L1 and DSCR1L2. The sequences of the respective protein products are of 198, 197 and 241 amino acids, respectively, and are very similar to the corresponding human proteins. The very broad expression pattern of the murine DSCR1 genes is similar to that of the human genes. Using a radiation hybrid panel, we mapped the murine DSCR1-like family members. The murine DSCR1 ortholog is located on the chromosome 16, in a region corresponding to that on human chromosome 21 just upstream of the Down syndrome candidate region. DSCR1L1 and DSCR1L2 murine genes are also located in chromosomal segments of chromosome 17 and 4, respectively, exactly corresponding to those containing the respective human homologs on chromosomes 6 and 1. Description of the mouse orthologs for DSCR1-like genes will allow knockout mice to be obtained for specific family members.

UniGene Tabulator: a full parser for the UniGene format

UNLABELLED UniGene Tabulator 1.0 provides a solution for full parsing of UniGene flat file format; it implements a structured graphical representation of each data field present in UniGene following import into a common database managing system usable in a personal computer. This database includes related tables for sequence, protein similarity, sequence-tagged site (STS) and transcript map interval (TXMAP) data, plus a summary table where each record represents a UniGene cluster. UniGene Tabulator enables full local management of UniGene data, allowing parsing, querying, indexing, retrieving, exporting and analysis of UniGene data in a relational database form, usable on Macintosh (OS X 10.3.9 or later) and Windows (2000, with service pack 4, XP, with service pack 2 or later) operating systems-based computers. AVAILABILITY The current release, including both the FileMaker runtime applications, is freely available at http://apollo11.isto.unibo.it/software/

Cysteine and tyrosine-rich 1 (CYYR1), a novel unpredicted gene on human chromosome 21 (21q21.2), encodes a cysteine and tyrosine-rich protein and defines a new family of highly conserved vertebrate-specific genes.

A novel human gene has been identified by in-depth bioinformatics analysis of chromosome 21 segment 40/105 (21q21.1), with no coding region predicted in any previous analysis. Brain-derived DNA complementary to RNA (cDNA) sequencing predicts a 154-amino acid product with no similarity to any known protein. The gene has been named cysteine and tyrosine-rich protein 1 gene (symbol cysteine and tyrosine-rich 1, CYYR1). The CYYR1 messenger RNA was found by Northern blot analysis in a broad range of tissues (two transcripts of 3.4 and 2.2 kb). The gene consists of four exons and spans about 107 kb, including a very large intron of 85.8 kb. Analysis of expressed sequence tags shows high CYYR1 expression in cells belonging to the amine precursor uptake and decarboxylation system. We also cloned the cDNA of the murine ortholog Cyyr1, which was mapped by a radiation hybrid panel on chromosome 16 within the region corresponding to that containing the respective human homolog on chromosome 21. Sequence and phylogenetic analysis led to identification of several genes encoding CYYR1 homologous proteins. The most prominent feature identified in the protein family is a central, unique cysteine and tyrosine-rich domain, which is strongly conserved from lower vertebrates (fishes) to humans but is absent in bacteria and invertebrates.

Segmental paralogy in the human genome: a large-scale triplication on 1p, 6p, and 21q.

Few cases of large-scale segmental paralogy have been reported in the human genome. We have identified a large (approximately 500 kb) segment on human chromosome (HC) 21 (21q22) that is triplicated on HC 1 (1p35) and HC 6 (6p12-21). We also identified a new member of CLIC (Chloride Intracellular Channel) family on 21q, namely CLIC6. All three segments appear to include three functional members of three different gene families: DSCR1-like (Down Syndrome Candidate Region 1-like), CLIC, and AML/Runt (Acute Myeloid Leukemia/Runt). Molecular evolution analysis shows a common evolutionary origin for the triplicated regions. This finding of a further large-scale genomic triplication that went undetected at previously systematic automated searches provides a new model for gene divergence study and underlines the need for new tools to effectively detect inter-chromosomal similarity. An algorithm to overcome current limitations is proposed.

Oxidative stress, poly(ADP)ribosylation and aging: in vitro studies on lymphocytes from normal and Down's syndrome subjects of different age and from patients with Alzheimer's dementia.

Free radicals are formed in the body as a consequence of aerobic metabolism. Cells have developed a variety of antioxidant systems, that include classical antioxidant enzymes (superoxide dismutase, glutathione peroxidase, and catalase) as well as nonenzymatic oxy-radicals scavengers (vitamin E, urea, s-carotene and some more recently described substances such as carnosine) (1). However, a certain fraction of active oxygen species escapes the cellular defence and may cause transient or permanent damage to cellular components. According to one of the most interesting theory of aging is the “free radical theory of aging”, proposed by D. Harman (2) more than thirty years ago, where oxidative damage has been suggested as a major cause of aging. One of the prediction of this theory is an age-related decrease of the efficiency of antioxidant defence mechanisms.

Cytochrome b of protozoan mitochondria: relationships between function and structure

1. The sensitivity of ubiquinol:cytochrome c reductase to its most powerful inhibitors has been characterized in mitochondria from three ciliate and two trypanosome protozoans and compared with that in mitochondria of animals and plants. 2. Mitochondria of ciliates, particularly those of Tetrahymena pyriformis, are resistant to antimycin. 3. Mitochondria of trypanosomes are quite resistant to stigmatellin, as they exhibit a 40-fold higher titer than that in ciliate or animals mitochondria. 4. Both ciliates and trypanosomes are highly resistant to myxothiazol. 5. Correlations have been drawn between the natural resistance of the protozoan mitochondria to antimycin, stigmatellin and myxothiazol and peculiar features in the structure of their apocytochrome b, on the basis of an accurate alignment of the sequences of this protein.