Correlation of HLA-Cw*06 allele frequency with some clinical features of psoriasis vulgaris in the population of northern Poland

Psoriasis is a common skin disease with a genetic background and significant human leukocyte antigen (HLA) associations. HLA-Cw6 is the most frequently described association, particularly with psoriasis of the early onset type. Few studies of its correlation with various psoriasis clinical phenotypes and severity of the disease have been published so far; none in the Polish population. In this study 78 patients with psoriasis vulgaris were evaluated clinically and subdivided according to the age of onset and the type of psoriasis. A system of disease severity evaluation was applied to each patient. All patients and the control group (70 unrelated persons) were typed for HLA-Cw*06. The results show that Cw*06 allele frequency was higher in psoriatics than in the control group. Our investigation confirms that the Cw*06 allele is positively associated with psoriasis vulgaris of the early onset type, with a positive family history and its more severe form.     

Genotoxicity of inhalation anaesthetics: DNA lesions generated by sevoflurane in vitro and in vivo

A moderate genotoxic activity of halothane and isoflurane applied as volatile anaesthetics has already been shown. The aim of this work was to estimate a potential genotoxicity of sevoflurane, introduced to clinical practice later than halothane and isoflurane. A genotoxic activity of all three compounds was estimated by using the comet assay in human peripheral blood lymphocytes (PBL) proliferating in vitro. We demonstrated that in contrast to the previously studied anaesthetics, sevoflurane did not induce any increase in DNA migration in the studied conditions. To estimate a genotoxic effect of a prolonged exposure to halogenated anaesthetics in vivo, PBL taken from operating room personnel (n = 29) were tested for DNA degradation and compared with those from a control non-exposed group (n = 20). No significant differences were detected between the groups. We conclude that sevoflurane does not have genotoxic properties, both in vitro and in vivo.     

Determination of new derivatives of genistein in culture media by liquid chromatography

Methods for determination of genistein and its four new analogues in culture media have been developed to support studies on their potential anticancer activities. The investigated compounds were extracted from the media using liquid-liquid extraction with appropriate solvent. After evaporation of organic solvents each of the dry extracts was reconstituted in appropriate mobile phase. Reversed-phase HPLC was applied to quantitative determining of tested compounds. The methods are specific, sensitive and technically simple. They were used to evaluate concentration level of investigated compounds in experiments with human promyelocytic leukemia cells (HL-60 cell line).     

Role of thiocyanate, bromide and hypobromous acid in hydrogen peroxide-induced apoptosis

We have previously reported that H₂O₂-induced apoptosis in HL-60 human leukemia cells takes place in the presence of chloride, requires myeloperoxidase (MPO), and occurs through oxidative reactions involving hypochlorous acid and chloramines. We now report that when chloride is replaced by the pseudohalide thiocyanate, there is little or no H₂O₂-induced apoptosis. Furthermore, thiocyanate inhibits H₂O₂-induced apoptosis when chloride is present at physiological concentrations, and this occurs at thiocyanate concentrations that are present in human serum and saliva. In contrast, bromide can substitute for chloride in H₂O₂-induced apoptosis, but results in a lower percent of the cells induced into apoptosis. Hypobromous acid is likely a short-lived intermediate in this H₂O₂/MPO/bromide apoptosis, and reagent hypobromous acid and bromamines induce apoptosis in HL-60 cells. We conclude that the physiologic concentrations of thiocyanate found in human plasma could modulate the cytototoxicity of H₂O₂ and its resulting highly toxic MPO-generated hypochlorous acid by competing with chloride for MPO. Furthermore, the oxidative products of the reaction of thiocyanate with MPO are relatively innocuous for human leukemic cells in culture. In contrast, bromide can support H₂O₂/MPO/halide apoptosis, but is less potent than chloride and it has no effect in the presence of physiological levels of chloride.     

Subcellular localization of Pseudomonas pyocyanin cytotoxicity in human lung epithelial cells

The Pseudomonas aeruginosa secretory product pyocyanin damages lung epithelium, likely due to redox cycling of pyocyanin and resultant superoxide and H(2)O(2) generation. Subcellular site(s) of pyocyanin redox cycling and toxicity have not been well studied. Therefore, pyocyanin's effects on subcellular parameters in the A549 human type II alveolar epithelial cell line were examined. Confocal and electron microscopy studies suggested mitochondrial redox cycling of pyocyanin and extracellular H(2)O(2) release, respectively. Pyocyanin decreased mitochondrial and cytoplasmic aconitase activity, ATP levels, cellular reduction of 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide, and mitochondrial membrane potential. These effects were transient at low pyocyanin concentrations and were linked to apparent cell-mediated metabolism of pyocyanin. Overexpression of MnSOD, but not CuZnSOD or catalase, protected cellular aconitase, but not ATP, from pyocyanin-mediated depletion. This suggests that loss of aconitase activity is not responsible for ATP depletion. How pyocyanin leads to ATP depletion, the mechanism of cellular metabolism of pyocyanin, and the impact of mitochondrial pyocyanin redox cycling on other cellular events are important areas for future study.     

Dynamics of subgenomic hepatitis C virus replicon RNA levels in Huh-7 cells after exposure to nucleoside antimetabolites

Treatment with antimetabolites results in chemically induced low nucleoside triphosphate pools and cell cycle arrest in exponentially growing cells. Since steady-state levels of hepatitis C virus (HCV) replicon RNA were shown to be dependent on exponential growth of Huh-7 cells, the effects of antimetabolites for several nucleoside biosynthesis pathways on cell growth and HCV RNA levels were investigated. A specific anti-HCV replicon effect was defined as (i). minimal interference with the exponential cell growth, (ii). minimal reduction in cellular host RNA levels, and (iii). reduction of the HCV RNA copy number per cell compared to that of the untreated control. While most antimetabolites caused a cytostatic effect on cell growth, only inhibitors of the de novo pyrimidine ribonucleoside biosynthesis mimicked observations seen in confluent replicon cells, i.e., cytostasis combined with a sharp decrease in replicon copy number per cell. These results suggest that high levels of CTP and UTP are critical parameters for maintaining the steady-state level replication of HCV replicon in Huh-7 cells.     

Proteome analysis reveals phosphorylation of ATP synthase beta -subunit in human skeletal muscle and proteins with potential roles in type 2 diabetes

Insulin resistance in skeletal muscle is a hallmark feature of type 2 diabetes. An increasing number of enzymes and metabolic pathways have been implicated in the development of insulin resistance. However, the primary cellular cause of insulin resistance remains uncertain. Proteome analysis can quantitate a large number of proteins and their post-translational modifications simultaneously and is a powerful tool to study polygenic diseases like type 2 diabetes. Using this approach on human skeletal muscle biopsies, we have identified eight potential protein markers for type 2 diabetes in the fasting state. The observed changes in protein expression indicate increased cellular stress, e.g. up-regulation of two heat shock proteins, and perturbations in ATP (re)synthesis and mitochondrial metabolism, e.g. down-regulation of ATP synthase beta-subunit and creatine kinase B, in skeletal muscle of patients with type 2 diabetes. Phosphorylation appears to play a key, potentially coordinating role for most of the proteins identified in this study. In particular, we demonstrated that the catalytic beta-subunit of ATP synthase is phosphorylated in vivo and that the levels of a down-regulated ATP synthase beta-subunit phosphoisoform in diabetic muscle correlated inversely with fasting plasma glucose levels. These data suggest a role for phosphorylation of ATP synthase beta-subunit in the regulation of ATP synthesis and that alterations in the regulation of ATP synthesis and cellular stress proteins may contribute to the pathogenesis of type 2 diabetes.     

A cross-platform public domain PC image-analysis program for the comet assay

The single-cell gel electrophoresis, also known as the comet assay, has gained wide-spread popularity as a simple and reliable method to measure genotoxic and cytotoxic effects of physical and chemical agents as well as kinetics of DNA repair. Cells are generally stained with fluorescent dyes. The analysis of comets--damaged cells which form a typical comet-shaped pattern--is greatly facilitated by the use of a computer image-analysis program. Although several image-analysis programs are available commercially, they are expensive and their source codes are not provided. For Macintosh computers a cost-free public domain macro is available on the Internet. No ready for use, cost-free program exists for the PC platform. We have, therefore, developed such a public domain program under the GNU license for PC computers. The program is called CASP and can be run on a variety of hardware and software platforms. Its practical merit was tested on human lymphocytes exposed to gamma-rays and found to yield reproducible results. The binaries for Windows 95 and Linux, together with the source code can be obtained from: http://www.casp.of.pl.     

A novel mutation A1270G of the EDA1 gene causing Tyr343Cys substitution in ectodysplasin-A in a family with anhidrotic ectodermal dysplasia

The structure of the EDA1 gene was investigated in a patient with anhidrotic ectodermal dysplasia. Sequence analysis revealed a novel A1270G transition in exon 9 of the EDA1 gene in the patient and his uncle, whereas the patient's mother and grandmother were heterozygotes. This mutation resulted in Tyr343Cys substitution in the extracellular domain of the EDA1 gene product - ectodysplasin-A. The additional Cys343 was located between Cys332 and Cys346 and formed with Cys352 a cluster of four closely situated residues that could potentially form disulfide bonds. This mutation might affect the tertiary structure of the receptor-binding domain of ectodysplasin-A and precipitate the clinical symptoms of anhidrotic ectodermal dysplasia.