Replication timing of human telomeric DNA and other repetitive sequences analyzed by fluorescence in situ hybridization and flow cytometry.

The replication timing of telomeres seems to differ between species. Yeast telomeres are late replicating, whereas limited data from very few human cell lines have indicated telomere replication throughout S phase. In the present study a series of permanent cell lines and patient samples was investigated using a flow cytometric approach for telomere length determination based on in situ hybridization using peptide nucleic acid probes and DNA staining. This method permits selective analysis of cells in specific phases of the cell cycle without perturbation of the cell cycle machinery. The timing of replication of telomeric C(3)TA(2) and T(2)AG(3) repeats was found to differ between individual samples and could precede or be concomitant with the replication of bulk DNA. Replication of the T(2)AG(3) strand seemed to occur somewhat later than that of the C(3)TA(2) strand in some samples. (GTG)(n) and other repetitive sequences generally showed a replication pattern similar to that of the bulk of DNA with slightly individual differences, whereas centromeric DNA repeats consistently replicated within a short time frame in late S phase. The apparent variability in replication timing seen for telomeric DNA might suggest individual differences in firing of replication origins.

Immediate and long term effects of weight reduction in obese people with asthma: randomised controlled study.

OBJECTIVE: To investigate the influence of weight reduction on obese patients with asthma. DESIGN: Open study, two randomised parallel groups. SETTING: Private outpatients centre, Helsinki, Finland. PARTICIPANTS: Two groups of 19 obese patients with asthma (body mass index (kg/m(2)) 30 to 42) recruited through newspaper advertisements. INTERVENTION: Supervised weight reduction programme including 8 week very low energy diet. MAIN OUTCOME MEASURES: Body weight, morning peak expiratory flow (PEF), forced vital capacity (FVC), forced expiratory volume in one second (FEV(1)); and also asthma symptoms, number of acute episodes, courses of oral steroids, health status (quality of life). RESULTS: At the end of the weight reducing programme, the participants in the treatment group had lost a mean of 14.5% of their pretreatment weight, the controls 0.3%. The corresponding figures after one year were 11.3% and a weight gain of 2.2%. After the 8 week dieting period the difference in changes in percentage of predicted FEV(1) from baseline in the treatment and control groups was 7.2% (95% confidence interval 1.9% to 12.5%, P=0. 009). The corresponding difference in the changes in FVC was 8.6% (4. 8% to 12.5%, P<0.0001). After one year the differences in the changes in the two groups were still significant: 7.6% for FEV(1) (1. 5% to 13.8%, P=0.02) and 7.6% for FVC (3.5% to 11.8%, P=0.001). By the end of the weight reduction programme, reduction in dyspnoea was 13 mm (on a visual analogue scale 0 mm to 100 mm) in the treatment group and 1 mm in the control group (P=0.02). The reduction of rescue medication was 1.2 and 0.1 doses respectively (P=0.03). After one year the differences in the changes between the two groups were -12 for symptom scores (range -1 to -22, P=0.04) and -10 for total scores (-18 to -1, P=0.02). The median number of exacerbations in the treatment group was 1 (0-4) and in the controls 4 (0-7), P=0.001. CONCLUSION: Weight reduction in obese patients with asthma improves lung function, symptoms, morbidity, and health status.

Telomere analysis by fluorescence in situ hybridization and flow cytometry.

Determination of telomere length is traditionally performed by Southern blotting and densitometry, giving a mean telomere restriction fragment (TRF) value for the total cell population studied. Fluorescence in situ hybridization (FISH) of telomere repeats has been used to calculate telomere length, a method called quantitative (Q)-FISH. We here present a quantitative flow cytometric approach, Q-FISHFCM, for evaluation of telomere length distribution in individual cells based on in situ hybridization using a fluorescein-labeled peptide nucleic acid (PNA) (CCCTAA)3probe and DNA staining with propidium iodide. A simple and rapid protocol with results within 30 h was developed giving high reproducibility. One important feature of the protocol was the use of an internal cell line control, giving an automatic compensation for potential differences in the hybridization steps. This protocol was tested successfully on cell lines and clinical samples from bone marrow, blood, lymph nodes and tonsils. A significant correlation was found between Southern blotting and Q-FISHFCMtelomere length values ( P = 0.002). The mean sub-telomeric DNA length of the tested cell lines and clinical samples was estimated to be 3.2 kbp. With the Q-FISHFCMmethod the fluorescence signal could be determined in different cell cycle phases, indicating that in human cells the vast majority of telomeric DNA is replicated early in S phase.

Expression of activation markers CD23 and CD69 in B-cell non-Hodgkin's lymphoma.

The activation associated proteins CD23 and CD69 are expressed on cells of different lineages upon mitogenic stimulation. CD23 is a well characterized multifunctional protein in lymphocyte development recognized as a diagnostic marker for chronic lymphocytic leukaemia. CD69 is one of the earliest markers expressed after activation of T cells, but its function is unclear. The aim of this study was to investigate the expression of these antigens in B-cell non-Hodgkin's lymphomas (NHL) in relation to clinical behaviour. Ninety samples from 84 patients with NHL of B cell type were studied for the expression of CD23 and CD69 in CD20+ B cells by flow cytometric dual parameter analysis. In individual lymphomas the CD23 and CD69 antigens showed an "on or off" pattern with most or very few cells positive for each antigen. The CD23 antigen was expressed in 23 of 53 (43%) indolent lymphomas and in 2 of 37 (5%) aggressive cases. Most indolent lymphomas (81%) and about half the aggressive cases (53%) expressed the CD69 antigen. Thus, both markers were associated with indolent type. CD23 expression correlated with chronic lymphocytic leukaemia subtype and CD69 expression with male gender, advanced stage, newly diagnosed lymphoma and shorter survival.