Impact of genetic variations of the CYP1A1, GSTT1, and GSTM1 genes on the risk of coronary artery disease

Carcinogenic and toxic molecules produce DNA adducts that contribute to the development of atherosclerosis. Genetic polymorphisms of xenobiotic-detoxified enzymes, which control the level of DNA adducts, may affect both enzymatic activity and individual susceptibility to coronary artery disease (CAD). In this study we investigated the effects of genetic polymorphisms of the CYP1A1*2C, GSTT1, and GSTM1 enzymes on CAD risk in a Turkish population. Genotypes were determined for 132 CAD patients and 151 healthy controls by the polymerase chain reaction/restriction fragment length polymorphism method. There were no significant differences between patients and controls in terms of CYP1A1, GSTT1, and GSTM1 genotypes. Analysis of the possible interactions between the genotypes, after adjustment for the risk factors, demonstrated that individuals carrying CYP1A1 variant GSTT1 null genotypes had an 8.907-fold increased CAD risk compared to their wild status (p<0.05). We suggest that genetic polymorphisms of xenobiotic-metabolizing enzymes could play an important role in CAD. Therefore, CYP1A1 and GSTM1 polymorphisms should be considered as important parameters for the prediction of CAD.     

SNAP-tag technology mediates site specific conjugation of antibody fragments with a photosensitizer and improves target specific phototoxicity in tumor cells

Cancer cells can be killed by photosensitizing agents that induce toxic effects when exposed to nonhazardous light, but this also causes significant damage to surrounding healthy cells. The specificity of photodynamic therapy can be increased by conjugating photosensitizing agents to antibodies and antibody fragments that bind specifically to tumor cell antigens. However, standard conjugation reactions produce heterogeneous products whose targeting specificity and spectroscopic properties can be compromised. In this study, we used an antibody fragment (scFv-425) that binds to the epidermal growth factor receptor (EGFR) as a model to investigate the use of SNAP-tag fusions as an improved conjugation strategy. The scFv-425-SNAP-tag fusion protein allowed the specific conjugation of a chlorin e6 photosensitizer modified with O(6)-benzylguanine, generating a homogeneous product that was delivered specifically to EGFR(+) cancer cells and resulted in significant, tumor cell-specific cytotoxicity. The impact of our results on the development of photodynamic therapy is discussed.     

Effect of stress-induced lipid peroxidation on functions of rat peritoneal macrophages

The aim of the present study was to investigate the effects of stress-induced lipid peroxidation on macrophages' functions. Animals were subjected to 4 h immobilization at 4 degrees C in restraining devices. The peritoneal macrophages obtained from rats exposed to cold and restraint stress exhibited an increase in lipid peroxidation and a decline of chemotaxis and phagocytosis compared with control rats. After supplementation with vitamin E, the increment in thiobarbituric acid reactive substances (TBARS) content as the oxidative stress marker and the decline of chemotaxis and phagocytosis in peritoneal macrophages observed during cold-restraint stress was significantly removed. No significant change in catalase activity of peritoneal macrophages was observed in groups exposed to cold-restraint stress and treated with vitamin E. These findings indicate that phagocytic and chemotactic capacities of peritoneal macrophages are decreased by cold-restraint stress and this effect of stress may be related to lipid peroxidation.     

First Homo erectus from Turkey and implications for migrations into temperate Eurasia

Remains of fossil hominins from temperate regions of the Old World are rare across both time and space, but such specimens are necessary for understanding basic issues in human evolution including linkages between their adaptations and early migration patterns. We report here the remarkable circumstances surrounding the discovery of the first fossil hominin calvaria from Turkey. The specimen was found in the Denizli province of western Turkey and recovered from within a solid block of travertine stone as it was being sawed into tile-sized slabs for the commercial natural stone building market. The new specimen fills an important geographical and temporal gap and displays several anatomical features that are shared with other Middle Pleistocene hominins from both Africa and Asia attributed to Homo erectus. It also preserves an unusual pathology on the endocranial surface of the frontal bone that is consistent with a diagnosis of Leptomeningitis tuberculosa (TB), and this evidence represents the most ancient example of this disease known for a fossil human. TB is exacerbated in dark-skinned peoples living in northern latitudes by a vitamin D deficiency because of reduced levels of ultraviolet radiation (UVR). Evidence for TB in the new specimen supports the thesis that reduced UVR was one of the many climatic variables presenting an adaptive challenge to ancient hominins during their migration into the temperate regions of Europe and Asia.     

Stability of a 24-meric homopolymer: comparative studies of assembly-defective mutants of Rhodobacter capsulatus bacterioferritin and the native protein

The stability of Rhodobacter capsulatus bacterioferritin, a 24-meric homopolymer, toward denaturation on variation in pH and temperature, and increasing concentrations of urea and guanidine.HCl was investigated with native PAGE, and CD and fluorescence spectroscopies. With temperature and urea, the wild-type protein denatured without discernible intermediates in the equilibrium experiments, but with guanidine.HCl (Gnd.HCl) one or more intermediate species were apparent at relatively low Gnd.HCl concentrations. Dissociated subunit monomers, or aggregates smaller than 24-mers containing the high alpha-helical content characteristic of the native protein were not obtained at any pH without a high proportion of the 24-mer being present, and taken together with the other denaturation experiments and the construction of stable subunit dimers by site-directed mutagenesis, this observation indicates that folding of the bacterioferritin monomer could be coupled to its association into a dimer. Glu 128 and Glu 135 were replaced by alanine and arginine in a series of mutants to determine their role in stabilizing the 24-meric oligomer. The Glu128Ala, Glu135Ala and Glu135Arg variants retained a 24-meric structure, but the Glu128Ala/Glu135Ala and Glu128Arg/Glu135Arg variants were stable subunit dimers. CD spectra of the Glu135Arg, Glu128Ala/Glu135Ala, and Glu128Arg/Glu135Arg variants showed that they retained the high alpha-helical content of the wild-type protein. The 24-meric Glu135Arg variant was less stable than the wild-type protein (T(m), [Urea](50%) and [Gnd.HCl](50%) of 59 degrees C, 4.9 M and 3.2 M compared with 73 degrees C, approximately 8 M and 4.3 M, respectively), and the dimeric Glu128Arg/Glu135Arg variant was less stable still (T(m), [Urea](50%) and [Gnd.HCl](50%) of 43 degrees C, approximately 3.2 M and 1.8 M, respectively). The differences in stability are roughly additive, indicating that the salt-bridges formed by Glu 128 and Glu 135 in the native oligomer, with Arg 61 and the amino-terminal amine of neighboring subunits, respectively, contribute equally to the stability of the subunit assembly. The additivity and assembly states of the variant proteins suggest that the interactions involving Glu 128 and Glu 135 contribute significantly to stabilizing the 24-mer relative to the subunit dimer.     

The MukB-ParC Interaction Affects the Intramolecular,Not Intermolecular,Activities of Topoisomerase IV

Proper chromosome organization is accomplished through binding of proteins such as condensins that shape the DNA and by modulation of chromosome topology by the action of topoisomerases. We found that the interaction between MukB, the bacterial condensin, and ParC, a subunit of topoisomerase IV, enhanced relaxation of negatively supercoiled DNA and knotting by topoisomerase IV, which are intramolecular DNA rearrangements but not decatenation of multiply linked DNA dimers, which is an intermolecular DNA rearrangement required for proper segregation of daughter chromosomes. MukB DNA binding and a specific chiral arrangement of the DNA was required for topoisomerase IV stimulation because relaxation of positively supercoiled DNA was unaffected. This effect could be attributed to a more effective topological reconfiguration of the negatively supercoiled compared with positively supercoiled DNA by MukB. These data suggest that the MukB-ParC interaction may play a role in chromosome organization rather than in separation of daughter chromosomes.     

Protective Effect of Nigella sativa and Nigella damascena Fixed Oils Against Aflatoxin Induced Mutagenicity in the Classical and Modified Ames Test

The antioxidant and mutagenic/antimutagenic activities of the fixed oils from Nigella sativa (NSO) and Nigella damascena (NDO) seeds, obtained by cold press-extraction from the cultivar samples, were comparatively investigated for the first time. The antimutagenicity test was carried out using classical and modified Ames tests. The fatty acid composition of the fixed oils was characterized by gas chromatography–mass spectrometry (GC-MS) while the quantification of thymoquinone in the fixed oils was determined by UPC². The main components of the NSO and NDO were found to be linoleic acid, oleic acid, and palmitic acid. The results of the Ames test confirmed the safety of NSO and NDO from the viewpoint of mutagenicity. The results of the three antioxidant test methods were correlated with each other, indicating NDO as having a superior antioxidant activity, when compared to the NSO. Both NSO and NDO exhibited a significant protective effect against the mutagenicity induced by aflatoxin B1 in Salmonella typhimurium TA98 and TA100 strains. When microsomal metabolism was terminated after metabolic activation of the mycotoxin, a significant increase in antimutagenic activity was observed, suggesting that the degradation of aflatoxin B1 epoxides by these oils may be a possible antimutagenic mechanism. It is worthy to note that this is the first study to assess the mutagenicity of NSO and NDO according to the OECD 471 guideline and to investigate antimutagenicity of NDO in comparison to NSO against aflatoxin.