Telomere length regulates ISG15 expression in human cells.

Endogenous genes regulated by telomere length have not previously been identified in human cells. Here we show that telomere length regulates the expression of interferon stimulated gene 15 (ISG15, 1p36.33).

Mammalian cells contain a second nucleocytoplasmic hexosaminidase.

Some thirty years ago, work on mammalian tissues suggested the presence of two cytosolic hexosaminidases in mammalian cells; one of these has been more recently characterized in a recombinant form and has an important role in cellular function due to its ability to cleave beta-N-acetylglucosamine residues from a variety of nuclear and cytoplasmic proteins. However, the molecular nature of the second cytosolic hexosaminidase, named hexosaminidase D, has remained obscure. In the present study, we molecularly characterize for the first time the human and murine recombinant forms of enzymes, encoded by HEXDC genes, which appear to correspond to hexosaminidase D in terms of substrate specificity, pH dependency and temperature stability.

Haploinsufficiency of senescence evasion factor causes defects of hematopoietic stem cells functions.

The quality of hematopoietic stem cells (HSCs) is essentially defined by two characteristics, i.e., multilineage differentiation and self-renewal capacity.

Low telomerase activity: possible role in the progression of human medullary thyroid carcinoma.

Maintenance of telomere length has been reported to be an absolute requirement for unlimited growth of human tumour cells and in about 85% of cases, this is achieved by reactivation of telomerase, the enzyme that elongates telomeres. Only in rare cases, like in human medullary thyroid carcinomas (MTC), telomerase activity (TA) is low or undetectable; however, this does not limit tumours to become clinically significant. Here, we report that very low TA (below 5% of HEK293) observed in MTC cell strains derived from different patients, although not sufficient for immortalising the cells, is necessary for prolonging their replicative life span.

Contributions of DNA interstrand cross-links to aging of cells and organisms.

Impaired DNA damage repair, especially deficient transcription-coupled nucleotide excision repair, leads to segmental progeroid syndromes in human patients as well as in rodent models. Furthermore, DNA double-strand break signalling has been pinpointed as a key inducer of cellular senescence. Several recent findings suggest that another DNA repair pathway, interstrand cross-link (ICL) repair, might also contribute to cell and organism aging.

Identification of cultivation-independent markers of human endothelial cell senescence in vitro.

Human aging processes are regulated by many divergent pathways and on many levels. Thus, to understand such a complex system and define conserved mechanisms of aging, the use of cell culture-based models is a widespread practice. An often stated advantage of in vitro aging of primary cells is the high reproducibility compared to the much more intricate aging of organisms.

Early embryonic lethality of mice lacking the essential protein SNEV.

SNEV (Prp19, Pso4, NMP200) is a nuclear matrix protein known to be involved in pre-mRNA splicing, ubiquitylation, and DNA repair. In human umbilical vein endothelial cells, SNEV overexpression delayed the onset of replicative senescence. Here we analyzed the function of the mouse SNEV gene in vivo by employing homologous recombination in mice and conclude that SNEV is indispensable for early mouse development.

XT-II, the second isoform of human peptide-O-xylosyltransferase, displays enzymatic activity.

Peptide O-xylosyltransferase (EC 2.4.2.

Labelling of human adipose-derived stem cells for non-invasive in vivo cell tracking.

Human adipose-derived stem cells (ASC) can be expanded in an undifferentiated state or differentiated along the osteogenic, chondrogenic, adipogenic, myogenic, endothelial and neurogenic lineage. To test their in vivo and in situ regenerative potential, their fate needs to be traced after application in suitable defect models. Non-invasive imaging systems allow for real time tracking of labelled cells in the living animal.

Aging and the ubiquitinome: traditional and non-traditional functions of ubiquitin in aging cells and tissues.

Ubiquitination of endogenous proteins is one of the key regulatory steps of protein degradation followed by regulation of proteasome activity. During the last years evidence has increased that proteasome activity is decreased during the aging process in various model systems and that these changes might be causally related to aging and aging associated diseases. Since in most instances ubiquitination is the primary event in target selection, the system of ubiquitination and deubiquitination might be of similar importance.

Techniques in gerontology: cell lines as standards for telomere length and telomerase activity assessment.

The length of telomeres is believed to critically influence cellular aging processes and disease development. In order to reliably monitor telomere length and the corresponding cellular telomerase activity by optimized procedures, either based on flow cytometry or quantitative PCR technique, we here propose three commonly used cell lines, HEK293, K562 and TCL1301 as standards. In this contribution, efficient methods to determine mean telomere length of eukaryotic chromosomal DNA and determination of the corresponding telomeras activity are outlined.

SNEV overexpression extends the life span of human endothelial cells.

In a recent screening for genes down regulated in replicatively senescent human umbilical vein endothelial cells (HUVECs), we have isolated the novel protein SNEV. Since then SNEV has proven as a multifaceted protein playing a role in pre-mRNA splicing, DNA repair, and the ubiquitin/proteosome system. Here, we report that SNEV mRNA decreases in various cell types during replicative senescence, and that it is increased in various immortalized cell lines, as well as in breast tumors, where SNEV transcript levels also correlate with the survival of breast cancer patients.

SNEV is an evolutionarily conserved splicing factor whose oligomerization is necessary for spliceosome assembly.

We have isolated the human protein SNEV as downregulated in replicatively senescent cells. Sequence homology to the yeast splicing factor Prp19 suggested that SNEV might be the orthologue of Prp19 and therefore might also be involved in pre-mRNA splicing. We have used various approaches including gene complementation studies in yeast using a temperature sensitive mutant with a pleiotropic phenotype and SNEV immunodepletion from human HeLa nuclear extracts to determine its function.

Nuclear flow FISH: isolation of cell nuclei improves the determination of telomere lengths.

Understanding telomere biology is of utmost importance for aging and cancer research. An essential tool is the determination of telomere length, which traditionally is done by telomere restriction fragment analysis, a laborious and time consuming method. Therefore, large efforts have been made to establish alternative methods like flow FISH analysis.

Establishment of a strategy for the rapid generation of a monoclonal antibody against the human protein SNEV (hNMP200) by flow-cytometric cell sorting.

The screening for antigen-specific hybridoma cells with adequate production rates is still a time-, labour- and money-consuming procedure. A reduction in cell culture testing by specifically selecting those fused cells that produce antibody could therefore make hybridoma technology more attractive, even for small research groups or for newly discovered proteins at an early stage of research. Additional problems, such as the requirement to produce sufficient amounts of the unknown protein at a purity that allows specific immunisation of mice and testing of the resulting hybridoma clones, also need to be overcome.

Generation of an influenza A virus vector expressing biologically active human interleukin-2 from the NS gene segment.

Engineering of the influenza A virus NS1 protein became an attractive approach to the development of influenza vaccine vectors since it can tolerate large inserts of foreign proteins. However, influenza virus vectors expressing long foreign sequences from the NS1 open reading frame (ORF) are usually replication deficient in animals due to the abrogation of their NS1 protein function. In this study, we describe a bicistronic expression strategy based on the insertion of an overlapping UAAUG stop-start codon cassette into the NS gene, allowing the reinitiation of translation of a foreign sequence.

Comparison of early passage, senescent and hTERT immortalized endothelial cells.

The need for standardized experimental conditions to gain relevant and reproducible results has increased the demand for well characterized continuously growing cell lines that exhibit the characteristics of their normal counterparts. Immortalization of normal human cells by ectopic expression of the catalytic subunit of human telomerase (hTERT) has shown to result in highly differentiated cell lines. However, the influence of the increased telomerase activity on the protein expression profile was not investigated so far.

Interaction of U-box E3 ligase SNEV with PSMB4, the beta7 subunit of the 20 S proteasome.

Recognition of specific substrates for degradation by the ubiquitin-proteasome pathway is ensured by a cascade of ubiquitin transferases E1, E2 and E3. The mechanism by which the target proteins are transported to the proteasome is not clear, but two yeast E3s and one mammalian E3 ligase seem to be involved in the delivery of targets to the proteasome, by escorting them and by binding to the 19 S regulatory particle of the proteasome. In the present study, we show that SNEV (senescence evasion factor), a protein with in vitro E3 ligase activity, which is also involved in DNA repair and splicing, associates with the proteasome by directly binding to the beta7 subunit of the 20 S proteasome.

Screening for improved cell performance: selection of subclones with altered production kinetics or improved stability by cell sorting.

One of the major problems in the biotechnology industry is the selection of cell lines well suited for production of biopharmaceutical proteins. Usually, the most important selection criterion is the cell specific production rate. Nevertheless, a good producer cell line should have a number of additional advantageous properties, which allow the cell line to perform well in the type of bioreactor chosen for the process.

Distinct host range of influenza H3N2 virus isolates in Vero and MDCK cells is determined by cell specific glycosylation pattern.

Influenza A viruses were isolated in Vero, MDCK cells and chicken embryos. In contrast to MDCK-derived variants all H3N2 isolates obtained in Vero cells neither agglutinated chicken erythrocytes nor grew in chicken eggs. These host range differences of H3N2 Vero and MDCK isolates were noticed even in the absence of amino acid substitutions in the HA1 molecule.