Relative Human Telomere Length Quantification by Real-Time PCR.

Telomere measurement by quantitative PCR amplification is a well-known simple method to detect telomere length that involves large numbers of samples. The method has been developed by Cawthon in 2002 (Cawthon, Nucleic Acids Res 30:47e-47, 2002) and remains the most frequently used technique either in original or modified version. Telomere length is estimated by comparing the amount of telomere repeat amplification product (T) to a single copy gene (S) product. The T/S ratio correlates with the average telomere length. Cawthon suggested and recommended the use of 36B4 (RPLP0) as a single copy gene. However, Cawthon's suggestion was no longer considered a single copy gene and the gene was not suitable and appropriate for normalization.We thereby introduced a simple method for relative measurement of average human telomere length using quantitative real-time PCR. Our protocol was based on Cawthon's initial technique (Cawthon, Nucleic Acids Res 30:47e-47, 2002), modified by single-copy gene (SCG) primers and optimized.This technique is rapid, low cost, not demanding on DNA amount (or live cells), and can be used for a high-throughput screening and time monitoring.

Interleukin-1ß, interleukin-1 receptor antagonist, interleukin-6, interleukin-10, and tumor necrosis factor-α levels in CSF and serum in relation to the clinical diversity of Parkinson's disease.

Several parameters representing the clinical diversity of Parkinson's disease (PD), including severity, phenotypes, cognitive decline, anxiety and depression were analyzed to examine the link with interleukin-1ß (IL-1ß), the interleukin-1 receptor antagonist (IL-1RA), IL-6, IL-10, and tumor necrosis factor-α (TNFα) and also to determine the relationship between levels of these factors in serum and cerebrospinal fluid (CSF). Significantly elevated serum IL-1ß and IL-6 and reduced IL-1RA levels were found in the PD group. In CSF and serum, inflammatory factors behaved differently, with increased CSF TNFα indicating rapid PD progression, and increased IL-1ß in serum. A low level of IL-6 was associated with a longer duration of PD. Anxiety, depression, non-tremor phenotype and late-onset PD correlated with a high serum level of IL-10. The serum TNFα level was lower in PD patients with mild cognitive impairment compared to controls. Serum IL-1ß, IL-6 and IL-10 levels correlated with CSF markers.

Peptide regulation of gene expression and protein synthesis in bronchial epithelium.

INTRODUCTION: Some studies have shown that peptides have high treatment potential due to their biological activity, harmlessness, and tissue-specific action. Tetrapeptide Ala-Asp-Glu-Leu (ADEL) was effective on models of acute bacterial lung inflammation, fibrosis, and toxic lung damage in several studies. METHODS: We measured Ki67, Mcl-1, p53, CD79, and NOS-3 protein levels in the 1st, 7th, and 14th passages of bronchoepithelial human embryonic cell cultures. Gene expression of NKX2-1, SCGB1A1, SCGB3A2, FOXA1, FOXA2, MUC4, MUC5AC, and SFTPA1 was measured by real-time polymerase chain reaction. Using the methods of spectrophotometry, viscometry, and circular dichroism, we studied the ADEL-DNA interaction in vitro. RESULTS: Peptide ADEL regulates the levels of Ki67, Mcl-1, p53, CD79, and NOS-3 proteins in cell cultures of human bronchial epithelium in various passages. The strongest activating effect of peptide ADEL on bronchial epithelial cell proliferation through Ki67 and Mcl-1 was observed in "old" cell cultures. ADEL regulates the expression of genes involved in bronchial epithelium differentiation: NKX2-1, SCGB1A1, SCGB3A2, FOXA1, and FOXA2. ADEL also activates several genes, which reduced expression correlated with pathological lung development: MUC4, MUC5AC, and SFTPA1. Spectrophotometry, viscometry, and circular dichroism showed ADEL-DNA interaction, with a binding region in the major groove (N7 guanine). CONCLUSIONS: ADEL can bind to specific DNA regions and regulate gene expression and synthesis of proteins involved in the differentiation and maintenance of functional activity of the bronchial epithelium. Through activation of some specific gene expression, peptide ADEL may protect the bronchial epithelium from pulmonary pathology. ADEL also may have a geroprotective effect on bronchial tissue.

[The influence of age of donors on the ability of cells to homologous recombination DNA repair].

Old and young donors cells show different ability to homologous recombination (shown on the first stage--the chromosome transference) in vitro, that we suppose could be the reasons of the genome instability in aging. Homologous recombination, induced by X-radiation, is limited in cells taken from donors older than 70 years. Alpha-amanitin, the RNA-polymerize II repressor, in toxic doze, could induce the chromosome transference in the cells from all studied groups: from old and young donors and donors with repair process defect (with BRCA 1, 2 mutations). Summarized effect of X-radiation and alpha-amanitin does not increase the induction of the chromosome transference.

[Violation of the homologous chromosomes approchement as the answer to the small doses of roentgen irradiation].

Any violation of the homologous chromosome DNA repair leads to the genome instability, characteristic for hereditary syndromes and for aging cells. Using low doses of ionizing radiation (3-10 cGy) we have found any transference of the homologous centromere loci of the chromosome 1 (1q12) from the periphery to the cen- tre of the nucleus in the lymphocytes of young healthy donors. The same effect was found after any influence of RNA-polymerase inhibitor a-amanitine. Some changes in the chromatin structure during aging (70-80 years old patients) result in the difficulties in chromosome displacement, accompanied with any trouble in the appro- achement of the homologous chromosome loci as an answer to low doses of radiation.