An evolutionary change in telomere sequence motif within the plant section Asparagales had significance for telomere nucleoprotein complexes.

In association with a phylogenetic tree of Asparagales, our previous results showed that a distinct clade included plant species where the ancestral, Arabidopsis-type of telomeric repeats (TTTAGGG)n had been partially, or fully, replaced by the human-type telomeric sequence (TTAGGG)n. Telomerases of these species synthesize human repeats with a high error rate in vitro. Here we further characterize the structure of telomeres in these plants by analyzing the overall arrangement of major and minor variants of telomeric repeats using fluorescence in situ hybridization on extended DNA strand(s). Whilst the telomeric array is predominantly composed of the human variant of the repeat, the ancestral, Arabidopsis-type of telomeric repeats was ubiquitously observed at one of the ends and/or at intercalary positions of extended telomeric DNAs. Another variant of the repeat typical of Tetrahymena was observed interspersed in about 20% of telomerics. Micrococcal nuclease digestions indicated that Asparagales plants with a human-type of telomere have telomeric DNA organised into nucleosomes. However, unexpectedly, the periodicity of the nucleosomes is not significantly shorter than bulk chromatin as is typical of telomeric chromatin. Using electrophoretic mobility shift assays we detected in Asparagales plants with a human type of telomere a 40-kDa protein that forms complexes with both Arabidopsis- and human-type G-rich telomeric strands. However, the protein shows a higher affinity to the ancestral Arabidopsis-type sequence. Two further proteins were found, a 25-kDa protein that binds specifically to the ancestral sequence and a 15-kDa protein that binds to the human-type telomeric repeat. We discuss how the organisation of the telomere repeats in Asparagales may have arisen and stabilised the new telomere at the point of mutation.

Telomerase as a diagnostic and predictive marker in colorectal carcinoma.

In a search for molecular markers providing both informative diagnostics of malignant disease, and rational stratification of a therapeutic strategy to achieve optimal response in a given patient, we examined the possibility of using telomerase for this purpose in colorectal cancer. Telomerase, a ribonucleoprotein enzyme complex catalysing synthesis of chromosome ends (telomeres), has been known as an almost universal tumor marker but its predictive value has been found in only a limited number of malignant tumor types. Telomerase activity and expression of its catalytic subunit hTERT was determined in 82 surgical specimens from 41 patients (a sample of tumor tissue and of adjacent morphologically normal tissue was obtained from each patient). Telomerase activity was present in tumor samples from 34 (83%) patients, reaching an average value of 47.6 telomerase units (T.U.), while adjacent tissue specimens were either negative (in 25 (61%) patients), or slightly positive (in 16 (39%) patients) showing 1.5 T.U. on average. In tumor samples from patients without lymphatic node metastases (pN0), an average of 37.1 T.U was found. In contrast, in tumor samples from patients with lymphatic node involvement (pN1 or pN2) the average activity was significantly higher (60.2 T.U., p<0.05). In patients with distant metastases a tendency towards higher telomerase activity, although lacking statistical significance, could be observed. Among patients that obtained chemotherapy with 5-fluoruracil, those with low telomerase activity showed a tendency to chemosensitivity. Expression of hTERT was detected not only in samples showing telomerase activity, but also in a considerable portion of telomerase-negative samples either from the tumor or the adjacent normal tissue. We demonstrate that some of these apparent discrepancies may be attributed to differential splicing of hTERT mRNA. We conclude that TRAP assay for telomerase activity is more informative than the common testing for hTERT expression. Telomerase activity is useful both as a diagnostic as well as a predictive factor in colorectal cancer.

[Telomere analysis in tumor cells using in situ techniques]. / Analýza telomer nádorových bunek technikami in situ.

Stable telomere maintenance is essential for the indefinite cellular proliferation of germline and tumour cells. In most cases, telomere synthesis is performed by nucleoprotein enzyme complex of telomerase, that results in stabilisation of telomeres shortened to < or = 7 kb. Rarely, telomeres may be maintained via alternative (recombination-based) mechanism, which produces telomeres of heterogenous lengths (3-50 kb). Analysis of telomeres by in situ techniques, such as fluorescent in situ hybridisation (FISH) on metaphase spreads or on extended DNA fibres (fiber-FISH) and Primed in situ labelling (PRINS), enables to distinguish between these two mechanisms and to analyse individual telomeres in the given type of cells.

Application of a heated electrospray interface for on-line connection of the AAS detector with HPLC for detection of organotin and organolead compounds.

A heated electrospray interface that affords high sensitivity and long-term signal stability for AAS detection of metal-containing analytes in organic or organic-water solvents after HPLC separation is described. The vitreous body of the electrospray interface is externally heated above the boiling point of the solvent and quartz furnace AAS is used for detection. Interface working conditions were optimized with a full experimental design for the detection of tin- (tetramethyl-, tetraethyl-, tetrabutyl-, and tetrapentyltin, tributyltin chloride, dibutyltin dichloride, and butyltin trichloride) and lead- (tetraethyl- and tetraphenyllead) containing compounds in the column eluate. The heated electrospray interface enables use of a wide range of flow rates - from 50 to 1000 micro L min(-1). The measurement sensitivity and detection limit achieved were compared with those obtained by use of the thermospray interface and post-column conversion of the organotin compounds to gaseous hydrides. The detection limits for the low-molecular weight species of the homologous series (2.8+/-0.1 ng (140+/-5 ng mL(-1)) for tetramethyltin and 3.1+/-0.2 ng (155+/-10 ng mL(-1)) for tetraethyltin) were obtained approximately one order of magnitude lower than those obtained by use of the thermospray interface. With this HPLC-ES-QFAAS system the tributyltin content of BCR reference material 477, mussel tissue, was analyzed. This system was also applied to analysis of tetraethyllead in gasoline samples.

Telomerase activity and expression and telomere analysis in situ in the course of treatment of childhood leukemias.

Samples of blood and marrow from children with leukemia were assayed for telomerase activity and expression on the day of diagnosis and during the course of chemotherapy. A strong correlation between either variables and clinical response was observed in most patients. A unique case was observed in which telomerase activity was only moderately increased on diagnosis; it gradually increased in the course of therapy, and a subsequent decrease occurred only after application of intensified therapy. This patient did not respond to therapy, his disease progressed, and he finally died during intensified therapy. In another patient, analysis of telomere lengths using dideoxy-PRINS revealed a single telomere expansion on a long arm of chromosome 4, suggesting involvement of a telomerase-independent mechanism of telomere elongation.