Silvia Canaider

An estimation of the number of cells in the human body

Abstract Background: All living organisms are made of individual and identifiable cells, whose number, together with their size and type, ultimately defines the structure and functions of an organism. While the total cell number of lower organisms is often known, it has not yet been defined in higher organisms. In particular, the reported total cell number of a human being ranges between 1012 and 1016 and it is widely mentioned without a proper reference. Aim: To study and discuss the theoretical issue of the total number of cells that compose the standard human adult organism. Subjects and methods: A systematic calculation of the total cell number of the whole human body and of the single organs was carried out using bibliographical and/or mathematical approaches. Results: A current estimation of human total cell number calculated for a variety of organs and cell types is presented. These partial data correspond to a total number of 3.72 × 1013. Conclusions: Knowing the total cell number of the human body as well as of individual organs is important from a cultural, biological, medical and comparative modelling point of view. The presented cell count could be a starting point for a common effort to complete the total calculation.

Renaming the DSCR1/Adapt78 gene family as RCAN: regulators of calcineurin.

Kelvin J. A. Davies,* Gennady Ermak,* Beverley A. Rothermel, Melanie Pritchard, Joseph Heitman, Joohong Ahnn, Flavio Henrique-Silva, Dana Crawford, Silvia Canaider,** Pierluigi Strippoli,** Paolo Carinci,** Kyung-Tai Min, Deborah S. Fox, Kyle W. Cunningham, Rhonda Bassel-Duby, Eric N. Olson, Zhuohua Zhang, R. Sanders Williams, Hans-Peter Gerber,*** Merce Perez-Riba, Hisao Seo, Xia Cao, Claude B. Klee, Juan Miguel Redondo, Lois J. Maltais, Elspeth A. Bruford, Sue Povey, Jeffery D. Molkentin,**** Frank D. McKeon, Elia J. Duh, Gerald R. Crabtree,§§§§ Martha S. Cyert, Susana de la Luna, and Xavier Estivill

Transfection of annexin 1 in monocytic cells produces a high degree of spontaneous and stimulated apoptosis associated with caspase-3 activation

Transfection of the pre‐monomyelocytic U937 cell line with a plasmid coding for full‐length annexin 1 (ANX1, 347 amino acid) leads to cell death by promoting apoptosis. In addition, over‐expression of the N‐terminal and the first domain of the protein (144 amino acids, clone ANX1‐S), which does not contain the Ca2+ binding sites, gives susceptibility to cell apoptosis following activation by either 5 ng ml−1 tumour necrosis factor (TNF)‐α or 1 – 40 μg ml−1 etoposide. This was demonstrated by using the fluorescent labelled annexin V, cell cycle and nuclear staining analyses. Transfection with an empty plasmid (clone CMV) or with a plasmid carrying the cDNA antisense for ANX1 (clone ANX1‐AS) did not alter U937 cells to the degree of apoptosis promoted by either stimulant. Treatment of CMV U937 cells with TNF‐α increased ANX1 mRNA and protein expression in a time‐dependent manner, with maximal increases at 3 and 6 h, respectively. Clone ANX1‐S showed higher constitutive (more than 2 fold) and activated caspase‐3 activity, associated with higher phospholipase A2 (PLA2) activity (in the region of +50 – 100%), whereas expression of cytosolic PLA2 Bax and Bcl‐2 were similar in all cell clones, as determined by Western blotting. In conclusion, this study demonstrates a complex regulatory role of cell apoptosis for ANX1, at least with regards to cells of the myelo‐monocytic lineage.

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.

Increased apoptosis in U937 cells over-expressing lipocortin 1 (annexin I)

The potential involvement of endogenous lipocortin 1 in the process of cellular apoptosis, particularly in cells of the myelo-monocytic lineage, has been investigated. U937 cells were transfected either with an antisense or a sense DNA for lipocortin 1 and the stable clones 36.4AS clone (20-40% lower lipocortin 1 levels) and 15S (30% higher lipocortin 1 levels) were obtained. Cell apoptosis was induced by incubation with tumor necrosis factor-alpha: optimal responses were observed within a 24 h incubation period at a 5 ng/ml concentration. Apoptosis was assessed both morphologically, by annexin V binding and cell cycle analysis with propidium iodide. Whilst no consistent difference was seen between wild type cells and clone 36.4AS, a higher incidence of apoptosis (ranging from +30% to + 60%) was observed in the 15S clone. Release of arachidonic acid from loaded cells was promoted by 24 h incubation with the cytokine, and a higher degree of release was measured in the 15S clone. These data indicate that endogenous intracellular lipocortin 1 is involved in the promotion of apoptosis in cells of the myelo-monocytic derivation.

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.

Characterization of human gene locus CYYR1: a complex multi-transcript system.

Cysteine/tyrosine-rich 1 (CYYR1) is a gene we previously identified on human chromosome 21 starting from an in-depth bioinformatics analysis of chromosome 21 segment 40/105 (21q21.3), where no coding region had previously been predicted. CYYR1 was initially characterized as a four-exon gene, whose brain-derived cDNA sequencing predicts a 154-amino acid product. In this study we provide, with in silico and in vitro analyses, the first detailed description of the human CYYR1 locus. The analysis of this locus revealed that it is composed of a multi-transcript system, which includes at least seven CYYR1 alternative spliced isoforms and a new CYYR1 antisense gene (named CYYR1-AS1). In particular, we cloned, for the first time, the following isoforms: CYYR1-1,2,3,4b and CYYR1-1,2,3b, which present a different 3′ transcribed region, with a consequent different carboxy-terminus of the predicted proteins; CYYR1-1,2,4 lacks exon 3; CYYR1-1,2,2bis,3,4 presents an additional exon between exon 2 and exon 3; CYYR1-1b,2,3,4 presents a different 5′ untranslated region when compared to CYYR1. The complexity of the locus is enriched by the presence of an antisense transcript. We have cloned a long transcript overlapping with CYYR1 as an antisense RNA, probably a non-coding RNA. Expression analysis performed in different normal tissues, tumour cell lines as well as in trisomy 21 and euploid fibroblasts has confirmed a quantitative and qualitative variability in the expression pattern of the multi-transcript locus, suggesting a possible role in complex diseases that should be further investigated.

Complexity of Bidirectional Transcription and Alternative Splicing at Human RCAN3 Locus

Human RCAN3 (regulator of calcineurin 3) belongs to the human RCAN gene family. In this study we provide, with in silico and in vitro analyses, the first detailed description of the human multi-transcript RCAN3 locus. Its analysis revealed that it is composed of a multigene system that includes at least 21 RCAN3 alternative spliced isoforms (16 of them identified here for the first time) and a new RCAN3 antisense gene (RCAN3AS). In particular, we cloned RCAN3-1,3,4,5 (lacking exon 2), RCAN3-1a,2,3,4,5, RCAN3-1a,3,4,5, RCAN3-1b,2,3,4,5, RCAN3-1c,2,3,4,5, RCAN3-1c,2,4,5 and RCAN3-1c,3,4,5, isoforms that present a different 5′ untranslated region when compared to RCAN3. Moreover, in order to verify the possible 5′ incompleteness of previously identified cDNA isoforms with the reference exon 1, ten more alternative isoforms were retrieved. Bioinformatic searches allowed us to identify RCAN3AS, which overlaps in part with exon 1a, on the opposite strand, for which four different RCAN3AS isoforms were cloned. In order to analyze the different expression patterns of RCAN3 alternative first exons and of RCAN3AS mRNA isoforms, RT-PCR was performed in 17 human tissues. Finally, analyses of RCAN3 and RCAN3AS genomic sequences were performed to identify possible promoter regions, to examine donor and acceptor splice sequences and to compare evolutionary conservation, in particular of alternative exon 1 or 1c - exon 2 junctions in different species. The description of its number of transcripts, of their expression patterns and of their regulatory regions can be important to clarify the functions of RCAN3 gene in different pathways and cellular processes.