DNA repair genetic polymorphisms and risk of colorectal cancer in the Czech Republic

Colorectal cancer represents a complex disease where susceptibility may be influenced by genetic polymorphisms in the DNA repair system. In the present study we investigated the role of nine single nucleotide polymorphisms in eight DNA repair genes on the risk of colorectal cancer in a hospital-based case-control population (532 cases and 532 sex- and age-matched controls). Data analysis showed that the variant allele homozygotes for the Asn148Glu polymorphism in the APE1 gene were at a statistically non-significant increased risk of colorectal cancer. The risk was more pronounced for colon cancer (odds ratio, OR: 1.50; 95% confidence interval, CI: 1.01-2.22; p=0.05). The data stratification showed increased risk of colorectal cancer in the age group 64-86 years in both individuals heterozygous (OR: 1.79; 95% CI: 1.04-3.07; p=0.04) and homozygous (OR: 2.57; 95% CI: 1.30-5.06; p=0.007) for the variant allele of the APE1 Asn148Glu polymorphism. Smokers homozygous for the variant allele of the hOGG1 Ser326Cys polymorphism showed increased risk of colorectal cancer (OR: 4.17; 95% CI: 1.17-15.54; p=0.03). The analysis of binary genotype combinations showed increased colorectal cancer risk in individuals simultaneously homozygous for the variant alleles of APE1 Asn148Glu and hOGG1 Ser326Cys (OR: 6.37; 95% CI: 1.40-29.02; p=0.02). Considering the subtle effect of the DNA repair polymorphisms on the risk of colorectal cancer, exploration of gene-gene and gene-environmental interactions with a large sample size with sufficient statistical power are recommended.     

Oxalic acid stabilizes dopamine, serotonin, and their metabolites in automated liquid chromatography with electrochemical detection

Use of antioxidative agents is required in automated LC assay of microdialysis samples, due to rapid degradation of the monoamine neurotransmitters and their metabolites. Addition of oxalic acid prevented degradation of dopamine, serotonin, 3,4-dihydroxyphenylacetic acid, homovanillic acid and 5-hydroxyindoleacetic acid efficiently: after a 24-h incubation at room temperature the decreases in peak heights were less than 10%. The long-term stability of the analytes, however, was still enhanced when acetic acid and -cysteine were included in the solution. Using this antioxidative solution, the monoamine neurotransmitters and their metabolites could be determined with an automated LC assay even at room temperature.     

Anxiety with panic disorder linked to chromosome 9q in Iceland

The results of a genomewide scan for genes conferring susceptibility to anxiety disorders in the Icelandic population are described. The aim of the study was to locate genes that predispose to anxiety by utilizing the extensive genealogical records and the relative homogeneity of the Icelandic population. Participants were recruited in two stages: (1) Initial case-identification by a population screening for anxiety disorders, using the Stamm Screening Questionnaire, was followed by aggregation into extended families, with the help of our genealogy database; and (2) those who fulfilled the diagnostic and family aggregation criteria underwent a more detailed diagnostic workup based on the Composite International Diagnostic Interview. Screening for anxiety in close relatives also identified additional affected members within the families. After genotyping was performed with 976 microsatellite markers, affected-only linkage analysis was done, and allele-sharing LOD scores were calculated using the program Allegro. Linkage analysis of 25 extended families, in each of which at least one affected individual had panic disorder (PD), resulted in a LOD score of 4.18 at D9S271, on chromosome 9q31. The intermarker distance was 4.4 cM on average, whereas it was 1.5 cM in the linked region as additional markers were added to increase the information content. The linkage results may be relevant not only to PD but also to anxiety in general, since our linkage study included patients with other forms of anxiety.     

Matrix-independent survival of human keratinocytes through an EGF receptor/MAPK-kinase-dependent pathway

Normal epithelial cells undergo apoptosis when they are denied contact with the extracellular matrix, in a process termed "anoikis." Conversely, malignant epithelial cells typically acquire anchorage independence, i.e., the capacity to survive and grow in the absence of matrix interaction. Here we asked the question whether anoikis is affected by signaling through the EGF receptor (EGFR). We focused on the EGFR because EGFR signaling is frequently deregulated in malignant epithelial cells. We demonstrate that EGFR activation markedly alleviated the requirement of matrix engagement for survival of primary and immortalized human keratinocytes in suspension culture. Protection of epithelial cells through EGFR activation against anoikis was associated with and required sustained MAPK phosphorylation during the early phase of suspension culture. Interestingly, high levels of MAPK phosphorylation were not only required for EGFR-mediated protection against anoikis but also occurred as a consequence of caspase activation at later stages of suspension culture. These results demonstrate that EGFR activation contributes to anchorage-independent epithelial cell survival and identify MAPK activation as an important mechanism in this process.     

Molecular and cellular alterations in tobacco smoke-associated larynx cancer

Tumours of head and neck belong to the most frequent types of cancer world-wide. In Poland, mortality from larynx cancer among males has been continuously increasing during the last decades up to 8.4 deaths per 100,000 men in 1993, which exceeds epidemiological records from other countries. The aetiology of laryngeal cancer is strongly associated with exposure to carcinogens present in tobacco smoke. The review describes a sequence of molecular and cellular events from carcinogenic exposure, DNA adduct formation, detection of mutations in the p53 gene, loss of heterozygosity (LOH) in chromosomal loci encoding the p53 and p16 genes, and loss of control of the cell cycle. The section concerning DNA adducts includes a discussion of the role of such confounders as exogenous exposure, the age and sex of the subject, and disease progression. The significance of genetic factors as individual risk determinants is discussed in relation to bleomycin-induced chromosome instability and in connection with the occurrence of defects in genes encoding detoxifying enzymes. The question concerning the substantial difference between men and women in larynx cancer morbidity and mortality remains open, even when the significantly higher adduct formation in male DNA compared with female material was taken into account. Preliminary experiments suggest a role of the frequently observed loss of the Y-chromosome.     

Examples of high-frequency EPR studies in bioinorganic chemistry

Low-temperature EPR spectroscopy with frequencies between 95 and 345 GHz and magnetic fields up to 12 T has been used to study metal sites in proteins or inorganic complexes and free radicals. The high-field EPR method was used to resolve g-value anisotropy by separating it from overlapping hyperfine couplings. The presence of hydrogen bonding interactions to the tyrosyl radical oxygens in ribonucleotide reductases were detected. At 285 GHz the g-value anisotropy from the rhombic type 2 Cu(II) signal in the enzyme laccase has its g-value anisotropy clearly resolved from slightly different overlapping axial species. Simple metal site systems with S>1/2 undergo a zero-field splitting, which can be described by the spin Hamiltonian. From high-frequency EPR, the D values that are small compared to the frequency (high-field limit) can be determined directly by measuring the distance of the outermost signal to the center of the spectrum, which corresponds to (2 S-1)* mid R: Dmid R: For example, D values of 0.8 and 0.3 cm(-1) are observed for S=5/2 Fe(III)-EDTA and transferrin, respectively. When D values are larger compared to the frequency and in the case of half-integer spin systems, they can be obtained from the frequency dependence of the shifts of g(eff), as observed for myoglobin in the presence ( D=5 cm(-1)) or absence ( D=9.5 cm(-1)) of fluoride. The 285 and 345 GHz spectra of the Fe(II)-NO-EDTA complex show that it is best described as a S=3/2 system with D=11.5 cm(-1), E=0.1 cm(-1), and g(x)= g(y)= g(z)=2.0. Finally, the effects of HF-EPR on X-band EPR silent states and weak magnetic interactions are demonstrated.     

Protein folding coupled to DNA binding in the catalytic domain of bacteriophage lambda integrase detected by mass spectrometry

Bacteriophage lambda integrase (lambda-Int) is the prototypical member of a large family of enzymes that catalyze site-specific DNA recombination via single-strand cleavage and the formation of a Holliday junction intermediate. Crystallographic and biochemical evidence indicate that substantial conformational change (i.e., folding) in the catalytic domain of the protein is required for substrate recognition and catalysis. We have examined the solution conformation of the catalytic domain (C170) in the absence and presence of a cognate "half-site" DNA oligonucleotide by electrospray ionization mass spectrometry, and circular dichroism and fluorescence spectroscopy. The distribution of ions in the positive ion electrospray mass spectrum of the free protein reveals the presence of three distinct species in solution, one corresponding to the folded protein, one to the unfolded protein, and one to a dimer. In the presence of DNA, ions are observed only for the protein-DNA complex and the folded form of the free protein. We therefore conclude that DNA binding stabilizes the global fold of the protein in a manner that is consistent with folding-coupled target recognition as a mechanism to control site-specific recombination. Furthermore, we find that inspection of the charge state distribution of ions in electrospray mass spectra provides a quick and effective means to identify conformational heterogeneity of proteins in solution and to investigate dynamic protein-nucleic acid interactions.     

Changed energy state and increased mitochondrial beta-oxidation rate in liver of rats associated with lowered proton electrochemical potential and stimulated uncoupling protein 2 (UCP-2) expression: evidence for peroxisome proliferator-activated receptor-alpha independent induction of UCP-2 expression

Lowering of plasma triglyceride levels by hypolipidemic agents is caused by a shift in the liver cellular metabolism, which become poised toward peroxisome proliferator-activated receptor (PPAR) alpha-regulated fatty acid catabolism in mitochondria. After dietary treatment of rats with the hypolipidemic, modified fatty acid, tetradecylthioacetic acid (TTA), the energy state parameters of the liver were altered at the tissue, cell, and mitochondrial levels. Thus, the hepatic phosphate potential, energy charge, and respiratory control coefficients were lowered, whereas rates of oxygen uptake, oxidation of pyridine nucleotide redox pairs, beta-oxidation, and ketogenesis were elevated. Moderate uncoupling of mitochondria from TTA-treated rats was confirmed, as the proton electrochemical potential (Delta(p)) was 15% lower than controls. The change affected the Delta(Psi) component only, leaving the (Delta)pH component unaltered, suggesting that TTA causes induction of electrogenic ion transport rather than electrophoretic fatty acid activity. TTA treatment induced expression of hepatic uncoupling protein 2 (UCP-2) in rats as well as in wild type and PPARalpha-deficient mice, accompanied by a decreased double bond index of the mitochondrial membrane lipids. However, changes of mitochondrial fatty acid composition did not seem to be related to the effects on mitochondrial energy conductance. As TTA activates PPARdelta, we discuss how this subtype might compensate for deficiency of PPARalpha. The overall changes recorded were moderate, making it likely that liver metabolism can maintain its function within the confines of its physiological regulatory framework where challenged by a hypolipemic agent such as TTA, as well as others.