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PubMed2002

Telomerase activity and hTERT mRNA expression can be heterogeneous and does not correlate with telomere length in soft tissue sarcomas.

In a previous study, we showed that telomerase activity (TA) and human telomerase reverse transcriptase (hTERT) mRNA expression were undetectable in benign mesenchymal lesions and low-grade soft tissue sarcomas (STSs), but detectable in about 50% of intermediate-/high-grade STSs. We wondered if this lack of TA or hTERT mRNA expression could be related to the tumor sample examined and if there was a relationship between the former 2 parameters and telomere length. Two separate tumor samples from 37 STSs were examined for telomerase activity, using the telomerase repeat amplification protocol (TRAP) assay and for hTERT mRNA expression, using RT-PCR. Telomere length was determined in each tumor sample, using the terminal restriction fragments (TRF) technique. Significant variations in telomere length, TA and hTERT mRNA expression between 2 samples of the same tumor were observed in 27%, 11% and 27% of STSs, respectively. Telomere length did not correlate with TA or hTERT mRNA expression. Despite great intratumoral heterogeneity in telomere length, short and long telomeres were more often seen in the low/intermediate-grade and high-grade STS categories, respectively. Few STSs that showed a TRF pattern suggestive of alternative lengthening of telomeres (ALT) may contain ALT subpopulations.

Adolescent↗
PubMed2005

Assessment of young children using the Achenbach System of Empirically Based Assessment (ASEBA).

After providing a brief review of three other approaches to assessment of preschool children (DSM-IV diagnoses, "Zero to Three" diagnoses, and temperament scales), this paper focuses on the Achenbach System of Empirically Based Assessment (ASEBA). The empirically based assessment paradigm provides user-friendly, cost-effective, reliable, and valid procedures for assessing children's behavioral/emotional problems from the perspectives of multiple informants. The ASEBA preschool forms, the Child Behavior Checklist for ages 1.5-5 (CBCL/1.5-5) and the Caregiver-Teacher Report Form (C-TRF), are usable by many different kinds of professionals in diverse settings. The CBCL/1.5-5 also includes the Language Development Survey (LDS), which provides a quick screen for delays in vocabulary and word combinations. The problem items of the CBCL/1.5-5 and the C-TRF are scored on both empirically based syndromes and DSM-oriented scales, which are normed on the same general population sample. Variations in children's functioning across contexts and interaction partners make it essential to obtain and integrate data from multiple sources. Therefore, ASEBA software provides side-by-side comparisons of item and scale scores from up to eight assessment forms per child. Clinical and research applications of ASEBA preschool forms are summarized in the paper, and strengths and limitations are discussed.

Child, Preschool↗
PubMed2006

Excitation UNFOLD (XUNFOLD) to improve the temporal resolution of multishot tailored RF pulses.

An extension of the "UNaliasing by Fourier encoding the Overlaps using the temporaL Dimension" (UNFOLD) method to the excitation domain (XUNFOLD) is presented to improve the temporal resolution of multishot tailored RF (TRF) pulses. Multishot three-dimensional TRF pulses were designed to produce a time series of images with periodically aliased excitation profiles. The XUNFOLD method is shown to remove the excitation profile aliasing from the dynamic imaging data by filtering in the temporal frequency dimension. The technique is demonstrated to improve the temporal resolution of simulated functional MRI (fMRI) activation in a time series of brain images.

Algorithms↗
PubMed2003

Measurement of telomere length on tissue sections using quantitative fluorescence in situ hybridization (Q-FISH).

Loss of telomere repeat sequences occurs after each cell division and telomere shortening has been implicated in cellular senescence. The measurement of telomere length might therefore assess the lifespan of a cell. The aim of this study was to set up and validate a technique enabling the assessment of telomere length on tissue sections. Quantitative fluorescence in situ hybridization (Q-FISH) with telomeric probes was performed on smears and sections from cell preparations or human tissues. The mean fluorescence intensity of telomere spots (FI/spot) was automatically quantified by image analysis. Telomeric restriction fragment (TRF) length was assessed by Southern blotting. There was a positive significant correlation between telomere length, as assessed by Q-FISH, and TRF length determined by Southern blotting in corresponding samples (p < 0.01, r = 0.6 for tissue and p < 0.01, r = 0.79 for cells). FI/spot was higher on smears than on sections, but pairwise comparison showed a significant correlation both for cells and for tissues (r = 0.77, p < 0.001 for cells and p < or = 0.01, r = 0.64 for tissue). Finally, since telomere length is expected to shorten with age, FI/spot was assessed in liver samples according to the age of patients: a negative correlation was demonstrated (r = 0.76, p < 0.01). Inter-assay variation was 7% for Q-FISH performed on tissue sections and 12% on touch preparations. This study shows that Q-FISH can be performed with confidence on fixed frozen tissue sections in order to assess telomere length. It is an easy, accurate, and reproducible in situ method for assessing telomeres in the context of cell type and tissue architecture.

Adult↗
PubMed2001

Induction of telomere shortening and replicative senescence by cryopreservation.

Cryopreservation can alter cellular function under certain conditions. In this report, we demonstrate the induction of cellular senescence after cells have been cryopreserved using a standard protocol. A retinal pigment epithelial cell line frozen at a specific freezing rate and subsequently thawed showed severely impaired proliferation compared to cells that were not cryopreserved. The induction of senescence was suggested by senescent associated beta-galactosidase activity and diminished bromo-deoxyuridine incorporation. A remarkable increase of single-strand DNA breaks in terminal restriction fragment (TRF) were found in cryopreserved cells immediately after thawing. The rate of mean TRF length shortening was accelerated after cryopreservation. Given this evidence, we hypothesize that cryopreservation may cause telomere shortening and cellular senescence under certain freezing conditions.

Bromodeoxyuridine↗
PubMed1995

Telomere shortening is associated with cell division in vitro and in vivo.

In humans, the amount of terminal (TTAGGG)n, telomeric DNA decreases during aging of various somatic cell types in vitro and in vivo. While the factors accounting for telomere shortening have not been thoroughly established, the inability of the DNA replication machinery to completely copy chromosomal termini (the "end replication problem") and the absence in somatic cells of telomerase, the enzyme that synthesizes telomeric DNA de novo, is a likely mechanism. One prediction of this hypothesis is that telomere shortening should be dependent on cell division. Thus we analyzed telomere length in actively dividing and quiescent cells in vitro and in vivo. In circular outgrowths of cultured human diploid fibroblasts (HDF), cells at the outer periphery had a significantly lower mean terminal restriction fragment (TRF) length (P = 0.011) and telomeric signal intensity (P = 0.024) than cells at the center. Also, the rate of telomere shortening over time for HDFs held quiescent was not statistically significant (m = -12 bp/day, P = 0.16) while that for serially passaged cells was significant (m = -34 bp/day, P = 0.017). To examine the rate of telomere shortening for quiescent cells in vivo, we measured mean TRF length in brain tissue from adult donors ranging in age from 32-75 years. No significant decrease was observed as a function of donor age (P = 0.087), in contrast to the shortening of telomere length that occurs during in vivo aging of mitotically active cells (P = 0.0001). These observations show that telomere shortening is largely, if not entirely, dependent on cell division and support the end replication problem as a mechanism for this process and the use of telomere length as a biomarker for replicative capacity.

Adult↗
PubMed1997

Chromosomal instability and telomere length variations during the life span of human fibroblast clones.

Growth characteristics, karyotype changes, and telomere length variations were analyzed during the life span of 12 anchorage-independent clones isolated from a xeroderma pigmentosum fibroblast strain. After an initial period of comparable active growth, all the clones showed a decline in the growth rate and finally entered a phase of replicative senescence; however, the number of population doublings and the time required to enter senescence varied among the clones. Repeated cytogenetic analyses during culture propagation showed the appearance of chromosome anomalies, mainly telomeric association (tas) and unbalanced translocations. In all the clones the percentage of abnormal mitoses increased with culture passage, but reached different levels (from less than 10% to about 100%). This finding indicates that the replicative block may be associated with differently altered cytogenetic patterns. Specific chromosome arms (5p, 16q, 19q, and 20q) were preferentially involved in tas, suggesting that alterations in chromosome ends may occur which predispose to fusion. In some clones it was possible to demonstrate the origin of marker chromosomes from the evolution of tas. Telomere length analysis by Southern blotting on DNA samples prepared from 7 clones and from the parental cell lines showed that the terminal restriction fragment (TRF) profiles were homogeneous in senescent parental cells and in the clones during the last part of their life in culture, regardless of the degree of karyotype abnormalities. The homogeneity of the TRF profiles supports the hypothesis of a critical telomere length at senescence.

Cellular Senescence↗
PubMed2000

Loss of chromosome 13 in cultured human vascular endothelial cells.

In the vascular endothelium of human beings, telomere length is negatively related while the frequency of aneuploidy is positively related to donor age. Both in culture and in vivo the frequency of aneuploidy increases as telomere length is shortened. In this study we explored the relation between telomere length and aneuploidy in cultured human umbilical vein endothelial cells (HUVEC) by: (a) karyotype analysis and fluorescent in situ hybridization (FISH), (b) measurement of the terminal restriction fragments (TRF), and (c) assessment of replicative senescence by the expression of beta-galactosidase. Of 8 HUVEC strains, 7 cell strains lost chromosome 13, as shown by metaphase analysis and FISH of interphase cells. Five strains gained chromosome 11. In addition, five HUVEC strains became hypotetraploid shortly after the loss of chromosome 13. The loss of chromosome 13 was observed as early as PD 20, when mean TRF length was greater than 9 kb and the percentage of cells positive for beta-galactosidase was relatively low. The almost uniform loss of chromosome 13 suggests that this unique type of aneuploidy of HUVEC is the result of a progressive expression of clones with survival advantage.

Aneuploidy↗
PubMed1998

Telomere length distribution and Southern blot analysis.

Southern blot analysis of terminal restriction fragments (TRFs) is the standard method for quantitative examination of telomere length distributions. Since TRFs contain a subtelomeric component, central parameters of the TRF distribution n(L) such as the arithmetic mean (M) or the median (Me) cannot be derived directly from Southern blot data, i.e. from the optical density distribution OD(L). Several estimates have been applied instead; the seeming arithmetic mean A, the "center of mass" C, and the positions of maximal (P) and half-maximal optical density (P(1/2)). We show that C> A> M for any non-truncated distributions n(L), and P> M> P1/2 for any symmetrical unimodal n(L). Symmetric appearance on a Southern blot, however, suggests positive skewness of n(L). Thus, a lognormal form of n(L) may be considered. Then, C> A> M> P=Me> P(1/2). Alternatively, a Weibull distribution may be assumed. The latter is compatible with negative feedback-regulation of the telomere lengths. Using the maximum likelihood method we compare these distributions with FISH-data on telomere lengths in different cell types. The fit of the lognormal distribution is clearly superior. Lognormal genesis may relate to telomere breakage and recombination. Truncation of the upper end of the TRF distribution is possible due to Southern blot artifacts. Thereby, the order of the estimates may change to P> C> A. Having minimal sensitivity to truncation, P seems to be the optimal choice. however, the variability of P is high since peakedness of OD(L) and DNA length resolution are inversely related.

Animals↗
PubMed1977

Immunohistochemical localization of thyrotropin-releasing factor in the rat median eminence.

Antibodies raised in rabbits against a synthetic preparation of thyrotropin-releasing factor (TRF) were used in association with the immunoperoxidase histochemical technique to localize TRF in the rat median eminence. Using coronal sections cut through the mid-arcuate region, specific immuno-reactive material was observed in the medial and lateral regions of the superficial layer of the external median eminence.

Animals↗
PubMed1984

Transcription in the trfA region of broad host range plasmid RK2 is regulated by trfB and korB.

Transcription at various points in the trf A region of broad host range plasmid RK2 has been analysed by measuring expression of the galK gene inserted at EcoRI sites introduced previously by TB1723 transposition mutagenesis. Rightward transcription (anti-clockwise on RK2) probably from a single promoter, proceeds across two open reading frames coding for a 13 kD polypeptide of unknown function, and the trf A gene, which provides a protein(s) essential for plasmid replication. This transcription is not auto-regulated by the products of either open reading frame and is also not subject to significant attenuation prior to the end of the trfA open reading frame. Leftward transcription appears to be directed by at least two well separated promoters, the more leftward being three to four times stronger than the more rightward. Rightward, but not leftward, transcription is repressed about 9-fold by the trfB locus of RK2 alone (so far not separable from the loci korA and korD) in trans while the combination of the korB and trfB loci in trans represses both rightward transcription (about 100-fold) and leftward transcription (the stronger activity by 10 to 15-fold). Regulation of these operons is therefore qualitatively different. The kilD locus in the trfA region, which is suppressed by korD (trfB) is thus probably part of the rightward (trfA) operon, while leftward transcription may represent the start of an operon containing kilB. The results suggest that RK2kor loci act by repressing transcription of kil loci and that the kil and kor control circuits may be part of an interlocking system of RK2 genes involved in replication and stable maintenance.

Chromosome Mapping↗
PubMed1986

Genetic variation in natural and laboratory populations of the marsupial Sminthopsis crassicaudata.

Gel electrophoresis of blood proteins has detected allelic variation at five loci (TRF, PGD, SOD, ADA, GPI) in a laboratory colony of the dasyurid marsupial Sminthopsis crassicaudata. Family data show no significant departures from Mendelian expectations. Analysis of blood from wild-caught progenitors of the colony revealed significant differences in gene frequency between groups of animals captured from different parts of southern and central Australia and showed that there are two major population clusters. These interpopulation differences are particularly marked at the TRF locus and indicate that the river Murray is a barrier for this species.

Adenosine Deaminase↗
PubMed1990

Children with chronic renal failure in Sweden 1978-1985.

A survey of chronic renal failure (CRF) in Swedish children was carried out for the period 1978-1985, using age-related cut-off levels for creatinine concentrations corresponding approximately to a glomerular filtration rate of 30 ml/min per 1.73 m2. The mean annual incidence of CRF was 6.9 and of terminal renal failure (TRF) 4.4/million children. The prevalence increased during the study period, for preterminal renal failure from 14.1 (1978) to 26.1 (1985) and for TRF from 12.4 to 16/million children. The main groups of primary renal disease were malformations (42%), hereditary disorders (27%), and glomerular diseases (14%), while pyelonephritis with vesico-ureteral reflux only made up 5%.

Adolescent↗
PubMed1990

The internal consistency and concurrent validity of a Spanish translation of the Child Behavior Checklist.

The Child Behavior Checklist's applicability to a Hispanic sample was assessed by an examination of the instrument's internal consistency and concurrent validity. The CBCL and TRF were administered to a community sample representative of children of Puerto Rico aged 4 to 16. Cronbach's alpha was used to assess the internal consistency of empirically derived scales. The relation of CBCL and TRF scores to clinical diagnosis, adaptive functioning, and need for services served as indicators of the concurrent validity of the instrument's Spanish version. The results indicate that the total behavior problem scores on the instruments are good continuous measures of maladjustment for children in Puerto Rico. A child with high values on the scales has a high probability of being classified as a case by a psychiatrist. High levels of internal consistency were found in most subscales. Only scales comprising low prevalence problems showed poor internal consistency.

Adolescent↗
PubMed1979

[Soluble mediators as a means of communication between lymphozytes in immune reactions (author's transl)].

Soluble mediators play an important role in the positive and negative regulation of immune reactions. This has been particularly well documented for T-B-cooperation in the humoral immune response to T-dependent antigens. T-helper cells produce a T-cell replacing Factor (TRF) upon mitogenic or antigenic stimulation. It provides the signal to antigen triggered B-cells to begin with active antibody synthesis and secretion and thus it is a positively regulating mediator. Additional functional and some structural characteristics of TRF are described. Antigen specific and non-specific suppressor factors seem to be involved in the negative regulation of immune responses. Thus, the communication between cells of the immune system is largely conducted via soluble signal substances.

Antibody Formation↗
PubMed1987

Study of the dynamics of Sertoli cell secretions in a new superfusion, two-compartment culture system.

A new superfusion, two-compartment culture system recently developed in our laboratory was used to investigate the dynamic changes in bidirectional secretion of transferrin (Trf) and androgen binding protein (ABP) by rat Sertoli cells (Sc) cultured for up to 12 d under various experimental conditions. The system is unique in that the cells are grown on porous substrate and can be superfused independently at the apical (A) and basal (B) surfaces. The Sc formed confluent monolayers with tight junctions and were highly polarized, morphologically resembling their normal appearance in vivo. The bidirectional secretion patterns (total amount and A:B ratio) of both Trf and ABP were affected by the addition of hormones (testosterone, 10(-6) M; follicle stimulating hormone, 0.1 microgram/ml; and fetal bovine serum 2%), but not by changes in the medium flow rate (0.8 to 3.2 ml/h). The superfusion, two-compartment culture system provides a very useful model for culture of polarized cell monolayers and for the study of bidirectional secretions under more "physiologic" conditions than those provided by static cultures.

Androgen-Binding Protein↗
PubMed2002

A naive accelerometer acting in the continuum range.

The space experiment TRAMP (Thermal Radiation Aspects of Migrating Particles) flown in 1999 onboard the mission Foton 12 sponsored by the European Space Agency (ESA), was conceived to reveal and measure a new kind of forces, named Thermal Radiation Forces (TRF). The experiment was dramatically disturbed by the occurrence of undesired convective motions due to the rotation of the spacecraft. Apart from that, corrosion occurred in some parts of the flight apparatus, resulting in the presence of gas bubbles inside the experimental liquid, completely compromising the results. Consequently, the experiment did not allow to reveal and/or to measure TRF, but it turned out to be useful in another way, as a very sensitive accelerometer, since the accelerations deduced from velocity measurements concurred with those measured by the Quasi-Steady Acceleration Measurement (QSAM) system.

Acceleration↗
PubMed2003

Composition and conservation of the telomeric complex.

The telomere is composed of telomeric DNA and telomere-associated proteins. Recently, many telomere-associated proteins have been identified, and various telomere functions have been uncovered. In budding yeast, scRap1 binds directly to telomeric DNA, and other telomere regulators (Sir proteins and Rif proteins) are recruited to the telomeres by interacting with scRap1. Cdc13 binds to the most distal end of the chromosome and recruits telomerase to the telomeres. In fission yeast and humans, TTAGGG repeat binding factor (TRF) family proteins bind directly to telomeric DNA, and Rap1 proteins and other telomere regulators are recruited to the telomeres by interacting with the TRF family proteins. Both organisms have Pot1 proteins at the most distal end of the telomere instead of a budding-yeast Cdc13-like protein. Therefore, fission yeast and humans have in part common telomeric compositions that differ from that of budding yeast, a result that suggests budding yeast has lost some telomere components during the course of evolution.

Humans↗