Comparing telomere length of sister chromatids in human lymphocytes using three-dimensional confocal microscopy

BACKGROUND: The length of the terminal sequences of linear chromosomes changes dynamically during cellular proliferation. A crucial element in the study of telomere-related regulation mechanisms is the ability to measure telomere lengths of individual chromosomes. Individual telomere lengths can be measured using digital imaging fluorescence microscopy-based techniques. We extended this method using confocal microscopy for the acquisition of three-dimensional (3D) images. Consequently, variations in measured signal intensities due to erroneous focusing are avoided. METHODS: We employed our 3D telomere sizing method to compare telomere lengths of sister chromatids within metaphase preparations from human lymphocytes. The samples were treated following a quantitative fluorescence in situ hybridization (Q-FISH) protocol using fluorescein isothiocyanate (FITC)-labeled telomeric peptidic nucleic acid (PNA) probes and propidium iodide (PI) counterstain. RESULTS: We demonstrated that the telomere lengths of two sister chromatids are not necessarily equal in human lymphocytes. Profound statistical analysis demonstrated significant differences in the distribution of the sister chromatid telomere lengths, but we were not able to prove a discrete distribution of telomere sister ratios. These telomere length differences were more apparent in older individuals. CONCLUSION: Whereas the majority of sister telomere pairs have equal lengths, surprisingly, a minority was significantly different in each individual studied. We are convinced that these observations are not linked to the methodology or the protocol applied. We suggest that a biological phenomenon might be involved.     

ECVAM's activities on biologicals

This paper summarises key activities initiated and the progress achieved between April 1993 and June 2002 in implementing the Three Rs in one of ECVAM's priority areas - the production and quality control of biologicals. These have included: organising nine key workshops; financially supporting and/or participating in a number of prevalidation and/or validation studies; financial contributions and sponsorship to relevant international workshops, symposia and conferences; and financial support for the compilation of manuals and expert reports, and training in test methods. The paper complements the papers of Hendriksen et al. and Cussler et al. included in these proceedings.     

Future activities: ECVAM and the quality control of biologicals

ECVAM's activities in the field of biologicals have contributed in many ways to the successful incorporation of Three Rs methods, as summarised elsewhere in these proceedings. The progress achieved is impressive, but large numbers of animals are still needed in order to meet the requirements stipulated by various regulations. ECVAM's activities in this area should therefore be continued and extended. Besides the well-established organisation of ECVAM workshops and contributions to conferences, further prevalidation and validation studies should be funded. In addition, studies on refinement, and training courses on validated and well-established Three Rs methods, could be initiated. There is a need for more communication and information exchange, especially between regulators and industry concerning the Three Rs. ECVAM could provide a suitable forum for such activities. An ECVAM Biologicals Task Force should be established in order to define a list of priorities.     

ECVAM's response to the changing political environment for alternatives: consequences of the European Union chemicals and cosmetics policies

The European Centre for the Validation of Alternative Methods (ECVAM) has restructured its services by directly targeting the animal tests that need to be replaced. In view of the short time-lines for making available and implementing validated methods, ECVAM is offering to steer the process by bringing together the inputs of stakeholders and encouraging the early involvement of regulators. In essence, steering groups formed by ECVAM senior staff, and complemented with external experts, will carry out the project management and will coordinate the various inputs.     

Genetic basis of mitochondrial function and morphology in Saccharomyces cerevisiae

The understanding of the processes underlying organellar function and inheritance requires the identification and characterization of the molecular components involved. We pursued a genomic approach to define the complements of genes required for respiratory growth and inheritance of mitochondria with normal morphology in yeast. With the systematic screening of a deletion mutant library covering the nonessential genes of Saccharomyces cerevisiae the numbers of genes known to be required for respiratory function and establishment of wild-type-like mitochondrial structure have been more than doubled. In addition to the identification of novel components, the systematic screen revealed unprecedented mitochondrial phenotypes that have never been observed by conventional screens. These data provide a comprehensive picture of the cellular processes and molecular components required for mitochondrial function and structure in a simple eukaryotic cell.     

Telomere biology in mammalian germ cells and during development

The development of an organism is a strictly regulated program in which controlled gene expression guarantees the establishment of a specific phenotype. The chromosome termini or so-called telomeres preserve the integrity of the genome within developing cells. In the germline, during early development, and in highly proliferative organs, human telomeres are balanced between shortening processes with each cell division and elongation by telomerase, but once terminally differentiated or mature the equilibrium is shifted to gradual shortening by repression of the telomerase enzyme. Telomere length is to a large extent genetically determined and the neonatal telomere length equilibrium is, in fact, a matter of evolution. Gradual telomere shortening in normal human somatic cells during consecutive rounds of replication eventually leads to critically short telomeres that induce replicative senescence in vitro and probably in vivo. Hence, a molecular clock is set during development, which determines the replicative potential of cells during extrauterine life. Telomeres might be directly or indirectly implicated in longevity determination in vivo, and information on telomere length setting in utero and beyond should help elucidate presumed causal connections between early growth and aging disorders later in life. Only limited information exists concerning the mechanisms underlying overall telomere length regulation in the germline and during early development, especially in humans. The intent of this review is to focus on recent advances in our understanding of telomere biology in germline cells as well as during development (pre- and postimplantation periods) in an attempt to summarize our knowledge about telomere length determination and its importance for normal development in utero and the occurrence of the aging and abnormal phenotype later on.     

In vivo relevance of citrullinated proteins and the challenges in their detection

Citrullination is a posttranslational modification of arginine. It plays both a physiological role, for instance during apoptosis and epigenetics, and a pathological role in cancer or diseases of the central nervous system. Most research on citrullination to date focuses on its role in auto-immune diseases such as multiple sclerosis and rheumatoid arthritis. In this context, the exact knowledge of citrullination sites in a protein can provide invaluable information about the etiological importance of these citrullinated proteins. However, few techniques exist that can accurately detect citrullination on the peptide level. This review aims to give an overview of the different methods available to date for the detection of citrullinated proteins and peptides. These include 2D-SDS-PAGE and immunodetection, as well as specific mass spectrometry (MS) approaches, both labeled and unlabeled. These MS approaches have been developed to pinpoint the exact location of citrullination on the peptide level. Improving the currently existing detection strategies while focusing on the role of citrullinated proteins will be invaluable to elucidate the importance of this posttranslational modification in vivo.