Milena Stevanovic

Telomere–associated chromosome fragmentation: applications in genome manipulation and analysis

Telomere–associated chromosome fragmentation (TACF) is a new approach for chromosome mapping based on the non–targeted introduction of cloned telomeres into mammalian cells. TACF has been used to generate a panel of somatic cell hybrids with nested terminal deletions of the long arm of the human X chromosome, extending from Xq26 to the centromere. This panel has been characterized using a series of X chromosome loci. Recovery of the end clones by plasmid rescue produces a telomeric marker for each cell line and partial sequencing will allow the generation of sequence tagged sites (STSs). TACF provides a powerful and widely applicable method for genome analysis, a general way of manipulating mammalian chromosomes and a first step towards constructing artificial mammalian chromosomes.

The cDNA sequence and chromosomal location of the human SOX2 gene

We have characterized a cDNA containing the coding region of a human SOX gene expressed in fetal brain. The cDNA is 1085 bp long, contains an open reading frame of 317 amino acids, and displays a high degree of similarity with the mouse Sox-2 gene. Human SOX2 has been local ized to the long arm of Chromosome (Chr) 3 in the region q26.3-27. The mammalian sex-determining gene, SRY, encodes a protein that includes a sequence motif known as the HMG-box (Sinclair et al. 1990; Goodfellow and LovellBadge 1993). This motif is responsible for the different DNA-binding activities of the SRY protein, and mutation analysis suggests that a functional HMG-box is required for sex determination (Harley et al. 1992). The HMG-box motif is found widely in proteins that bind to DNA including transcription factors, proteins that bind to DNA with limited sequence specificity, and non-sequence-specific chromatin proteins (Ner 1992). The mammalian genome contains a family of genes that are related to SRY in the region that encodes the HMGbox. These genes have been called SOX genes (SRY-related HMG-box genes). In the original report, four genes were identified as members of the mouse Sox gene family (Gubbay et al. 1990). These have been named Sox-1, Sox2, Sox-3, and Sox-4 (previously referred to as al, a2, a3, and a4). All these genes are expressed during embryonic development as well as in some adult tissues; Sox-1, Sox2, and Sox-3 are expressed at the highest levels in the developing nervous system (Collignon 1993). Subsequently, SOX genes have been identified in a wide variety of phylogenetically diverse organisms including mammals, birds, and insects. In most cases, the sequence information available is limited to the HMG-box (Griffiths 1991; Denny et al. 1992a; Goze et al. 1993; Wright et al. 1993; van de Wetering and Clevers 1993; Chardard et al. 1993), and only

Modulation of SOX2 and SOX3 gene expression during differentiation of human neuronal precursor cell line NTERA2

The SOX genes comprise a family of transcriptional regulators implicated in the control of nervous system development. The developing brain is the major site of expression of many Soxgenes. Sox2 and Sox3 genes are predominantly expressed in the immature, undifferentiated cells of the neural epithelium throughout the entire CNS. NTERA2 is a human embryonal carcinoma cell line that phenotypically represents undifferentiated, pluripotent embryonic stem cells. In the presence of retinoic acid, cells differentiate into mature neurons providing an in vitro model for studying human genes that promote and regulate neural differentiation.In this study it is shown for the first time that the retinoic acid-induced neuronal differentiation of NTERA2 cells is accompanied by down-regulation of SOX2 and up-regulation of SOX3 gene during early phases of induction. These data suggest that the effects of retinoic acid on neural differentiation of NTERA2 EC cells might be mediated by modulation of SOX2 and SOX3 gene expression.

Antioxidant and antiproliferative activity of chokeberry juice phenolics during in vitro simulated digestion in the presence of food matrix

Chokeberry juice was subjected to in vitro gastric digestion in the presence of food matrix in order to determine the changes in polyphenol content and antioxidant activity. Addition of food matrix immediately decreased the total phenolic content, anthocyanin content, DPPH scavenging activity as well as total reducing power by 36%, 90%, 45% and 44%, respectively. After in vitro digestion, total phenolic content, anthocyanin content and reducing power are slightly elevated, but they are still lower than in initial non-digested juice. The effect of digested juice on Caco-2 cells proliferation was also studied, and the reduction of proliferative rate by approximately 25% was determined. Our results suggested that although a large proportion of chokeberry phenolics undergo transformation during digestion they are still potent as antioxidant and antiproliferative agents.

PBX1 and MEIS1 up-regulate SOX3 gene expression by direct interaction with a consensus binding site within the basal promoter region

Sox3/SOX3 [SRY (sex determining region Y)-box 3] is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. We have previously reported characterization of the SOX3 promoter and demonstrated that the general transcription factors NF-Y (nuclear factor-Y), Sp1 (specificity protein 1) and USF (upstream stimulatory factor) are involved in transcriptional regulation of SOX3 promoter activity. In the present study we provide the first evidence that the TALE (three-amino-acid loop extension) transcription factors PBX1 (pre-B-cell leukaemia homeobox 1) and MEIS1 (myeloid ecotropic viral integration site 1 homologue) participate in regulating human SOX3 gene expression in NT2/D1 cells by direct interaction with the consensus PBX/MEIS-binding site, which is conserved in all mammalian orthologue promoters analysed. PBX1 is present in the protein complex formed at this site with nuclear proteins from uninduced cells, whereas both PBX1 and MEIS1 proteins were detected in the complex created with extract from RA (retinoic acid)-induced NT2/D1 cells. By functional analysis we also showed that mutations of the PBX1/MEIS1-binding sites resulted in profound reduction of SOX3 promoter responsiveness to RA. Finally, we demonstrated that overexpressed PBX1 and MEIS1 increased endogenous SOX3 protein expression in both uninduced and RA-induced NT2/D1 cells. With the results of the present study, for the first time, we have established a functional link between the TALE proteins, PBX1 and MEIS1, and expression of the human SOX3 gene. This link is of particular interest since both TALE family members and members of the SOX superfamily are recognized as important developmental regulators.

Improved transfection efficiency of cultured human cells

We simultaneously tested the transfection efficiency of NT2/D1 and HeLa cells with Lipofectamine™ (Life Technologies) and Effectene™ (Qiagen) transfection reagents using the pCH110 eukaryotic assay vector, which contains the lacZ reporter gene. Under our culture conditions for NT2/D1 and HeLa cells, Effectene™ transfection efficiency could be augmented by simply increasing the amount of plasmid DNA 1.5–3 times above the recommended concentration without any visible cytotoxicity. With the Lipofectamine™ reagent, optimal transfection efficiency was obtained for both cell lines within the recommended concentrations, but at the top of the range. The results indicate that optimization of the transfection process should include plasmid DNA concentrations above the levels suggested by the manufacturers, in order to accomplish the highest transfection efficiency.

Characterisation and mapping of the human SOX14 gene

SOX genes comprise a family of genes that are related to the mammalian sex determining gene SRY in the region that encodes the HMG-box domain responsible for the sequence-specific DNA-binding activity. SOX genes encode putative transcriptional regulators implicated in the decision of cell fates during development and the control of diverse developmental processes. We have cloned and characterised SOX14, a novel member of the human SOX gene family. Based on the HMG-box sequence, human SOX14 is a member of the B subfamily. SOX14 is expressed in human foetal brain, spinal cord and thymus, and like other members of the B subfamily, it might have a role in regulation of nervous system development. While other members of the B subfamily show similarity outside the HMG-box, the regions flanking the HMG box of the human SOX14 gene are unique. SOX14 has been mapped to human chromosome 3q22→ q23, close to the marker D3S1549. This location places SOX14 within a chromosome interval associated with two distinct syndromes that affect craniofacial development: Blepharophimosis-ptosis-epicantus inversus syndrome and Möbius syndrome.

Up‐regulation of the SOX3 gene expression by retinoic acid: characterization of the novel promoter‐response element and the retinoid receptors involved

Sox3/SOX3 gene is considered to be one of the earliest neural markers in vertebrates and it is implicated in the genetic cascades that direct brain formation. We have previously shown that early phases of differentiation and neural induction of NT2/D1 embryonal carcinoma cells by retinoic acid (RA) involve up‐regulation of the SOX3 gene expression. Here, we present identification of a novel positive regulatory promoter element involved in RA‐dependent activation of the SOX3 gene expression in NT2/D1 cells. This element represents a direct repeat 3‐like motif that directly interacts with retinoid X receptor (RXR) α in a sequence‐specific manner. It is capable of independently mediating the RA effect in a heterologous promoter context and its disruption caused significant reduction of RA/RXR transactivation of the SOX3 promoter. Furthermore, by using synthetic antagonists of retinoid receptors, we have shown for the first time, that RA‐induced SOX3 gene expression could be significantly down‐regulated by the synthetic antagonist of RXR. Also, this data showed that RXRs, but not RA receptors, are mediators of RA effect on the SOX3 gene up‐regulation in NT2/D1 cells. Presented data will be valuable for future investigation of SOX3 gene expression, not only in NT2/D1 model system, but also in diverse developmental, physiological and pathological settings.

Mycotherapy of Cancer: An Update on Cytotoxic and Antitumor Activities of Mushrooms, Bioactive Principles and Molecular Mechanisms of their Action

Mycotherapy is defined as the study of the use of extracts and compounds obtained from mushrooms as medicines or health-promoting agents. The present review updates the recent findings on anticancer/antitumor agents derived from mushroom extracts and their metabolites. The increasing number of studies in the past few years revealed mushroom extracts as potent antitumor agents. Also, numerous studies were conducted on bioactive compounds isolated from mushrooms reporting the heteropolysaccharides, β-glucans, α-glucans, proteins, complexes of polysaccharides with proteins, fatty acids, nucleoside antagonists, terpenoids, sesquiterpenes, lanostanoids, sterols and phenolic acids as promising antitumor agents. Also, molecular mechanisms of cytotoxicity against different cancer cell lines are discussed in this review. Findings with Antrodia camphorata and Ganoderma lucidium extracts and isolated compounds are presented, as being the most deeply studied previously.

The human SOX18 gene: cDNA cloning and high resolution mapping.

SOX genes comprise a family of genes that are related to the mammalian sex determining gene SRY and these genes play key roles during animal development. We report here cloning and characterisation of the human SOX18 gene. SOX18 gene is expressed in foetal brain as well as in a wide range of foetal and adult tissues indicating its function is not restricted to early development. Mapping analysis has revealed that SOX18 gene is located on human chromosome 20q13.3, 27.29 cR distal from the marker D20S173.