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INTRODUCTION Transforming growth factor β (TGF-β) is thought to be a vasoprotective cytokine. Numerous reports confirm its significance in blood and plaques. There is, however, a lack of information on the molecular mechanisms involving TGF-β in circulating inflammatory cells in atherogenesis. sThe aim of the study was to assess gene expression of TGF-β and its receptors in monocytes from patients with acute coronary syndromes (ACS) and the effect of standard treatment on the studied genes. MATERIAL AND METHODS The study was carried out in 32 patients with ACS and 15 healthy subjects. Gene expression of TGF-β and receptors TGF-βRI and TGF-βRII was evaluated on day 1 and 5 in the study group and once in controls. The number of mRNA copies isolated from monocytes was assessed by QRT-PCR. RESULTS Monocytes of ACS patients showed slightly elevated transcriptional activity of TGF-β1 and its receptors RI and RII genes (0.29 ±0.043 vs. 0.08 ±0.020, p = 0.05; 0.071 ±0.022 vs. 0.036 ±0.023, p < 0.05; 0.134 ±0.020 vs. 0.048 ±0.016, p < 0.05, respectively). After 5-day standard treatment modest reduction of TGF-βRI expression was observed. The studied genes' expression was unrelated to ejection fraction, myocardial necrosis markers, GRACE score, time from the onset of pain to percutaneous coronary intervention and angiographic findings. Among risk factors family history of CAD was associated with increased TGF-βRI expression. Moreover, the presence of 4 or more classic risk factors correlated with higher TGF-βRI expression. CONCLUSIONS Monocytes of ACS patients demonstrate overexpression of TGF-β1 and its receptors' genes. Five-day standard treatment downregulated the TGF-βRI gene but did not affect TGF-β1 and TGF-βRII.

Background: Inflammation plays a critical role in all stages of atherogenesis, including plaque destabilization leading to the rupture and local thrombosis, clinically manifested as unstable angina (UA) or myocardial infarction (MI). Recent data report enhanced expression of numerous pro-inflammatory genes in patients with acute coronary syndrome (ACS) both in plaque and in inflammatory cells. Kinins are peptides involved in vasodilation, vascular permeability, pain and inflammation. Their effects are mediated by two receptors, B1 and B2. As the role of kinins in ACS is not clear, the aim of the study was to assess the expression of the genes encoding kinin receptors in patients with ACS. Methods: The study was carried out on 40 patients with ACS and 10 age-matched healthy subjects (control (C)). To evaluate gene expression of B1 and B2 kinin receptors, total mRNA was extracted from peripheral blood mononuclear cells and the number of mRNA copies was assessed by quantitative reverse transcriptase–polymerase chain reaction. Results: In patients with MI and UA, the B1 receptor (B1R)/B2 receptor (B2R) ratio was inversed compared with healthy subjects (C group) (MI vs C: 1.54 ± 0.39 vs 0.36 ± 0.04; P

Cardiac syndrome X (CSX) is defined by typical chest pain, ST segment depression on ECG and normal coronary angiography. Pathology of CSX may involve microvascular dysfunction related to inflammation and abnormal pain sensitivity. Kinins are labile peptides participating in vasodilation, inflammation and pain. Their effects are mediated by two receptors: B1 and B2. The aim of the study was to assess gene expression of kinin receptors in peripheral blood mononuclear cells (PBMC) from patients with CSX.The study was carried out in 34 patients with cardiac syndrome X, 13 with unstable angina and ten healthy subjects. Total mRNA was extracted from PBMC and the number of mRNA copies was assessed by quantitive reverse transcriptase polymerase chain reaction.The study showed 7-fold higher transcriptional activity of B1R in CSX vs. control and 3.5 higher vs. UA. B2R expression was 2.5-fold higher in CSX group vs. control and UA, while in the letter two groups it was similar. Such disturbance in kinin signaling may participate in local vasoconstriction and may reflect disturbances in kinin signaling leading to nociceptive disturbances in these patients.

The purpose of this study was to examine the usefulness of electron paramagnetic resonance spectroscopy (EPR) to estimate zinc and copper ions biosorption from the environment by pigmented soil fungi Cladosporium cladosporioides. The existence of a low amount of pheomelanin, besides eumelanin, in C. cladosporioides samples was proved by the analysis of shape of their EPR spectra. Concentration of o-semiquinone free radicals in crude mycelium was 2.4x10(17) spin/g. Changes in free radicals system of C. cladosporioides cultured in the presence of Zn2+ and Cu2+ were analysed. Both magnetic and chemical interactions of zinc and copper ions with free radicals in C. cladosporioides melanin were found. Magnetically interacting diamagnetic Zn2+ ions increased the concentration of o-semiquinone free radicals in melanin existing in C. cladosporioides mycelium, whereas paramagnetic Cu2+ ions decreased this concentration. Chemical interactions of Zn2+ and Cu2+ ions decreased the free radical concentrations in C. cladosporioides melanin. Homogeneously distributed free radicals in C. cladosporioides melanin rise its activity in biosorption processes.

Among all species analyzed, the domestic pig seems to be the most appropriate organ donor for xenotransplantation. Porcine endogenous retroviruses (PERVs) are present in genomes of all pigs and are capable of infecting human cells in vitro thus posing a serious threat for xenotransplantation procedures. Despite the abundant distribution of PERVs integrated with porcine genome, the majority of PERV proviral DNA is not capable of expressing viral proteins unless seriously mutated. The aim of the study was to analyze PERV genome for mutations. The study was performed on blood samples from 146 pigs. Long-range polymerase chain reaction (Long-PCR) was performed with primer sets designed within long terminal repeats (LTRs). Long-PCR products of different molecular weights were obtained: 530 bp (33.1% of individuals), 580 bp (76.7%), 933 bp (100%), and 2900 bp (59.8%). Amplimers of 7200 bp were absent in 12.8% of individuals, indicating the lack of intact proviral DNA. Sequence analysis showed that most PERV proviral DNA was significantly mutated, thus suggesting the inability to express functional viral RNA; however, it cannot be ruled out that compensatory recombination processes could occur enabling replication of defective proviruses.

E. Buszmana,∗, B. Pilawa, M. Zdybel, D. Wrześniok, A. Grzegorczyk and T. Wilczok Department of Pharmaceutical Chemistry School of Pharmacy, Medical University of Silesia Jagiellonska 4, 41-200 Sosnowiec, Poland Department of Medical Physics School of Pharmacy, Medical University of Silesia Narcyzow 1, 41-200 Sosnowiec, Poland Department of Molecular Biology and Medical Genetics School of Pharmacy, Medical University of Silesia Narcyzow 1, 41-200 Sosnowiec, Poland

Bradykinin is a mediator of inflammation, responsible for pain, vasodilation, and capillary permeability. Bradykinin receptor 1 (B1R) and bradykinin receptor 2 (B2R) are G protein-coupled receptors that mediate kinin effects. The latter is constitutive and rapidly desensitized; the former is induced by inflammatory cytokines and resistant to densensitization. The distribution of bradykinin receptors in human intestinal tissue was studied in patients with inflammatory bowel disease (IBD), namely ulcerative colitis (UC) and Crohn's disease (CD). Both B2R and B1R proteins are expressed in the epithelial cells of normal and IBD intestines. B1R protein is visualized in macrophages at the center of granulomas in CD. B2R protein is normally present in the apexes of enterocytes in the basal area and intracellularly in inflammatory tissue. In contrast, B1R protein is found in the basal area of enterocytes in normal intestine but in the apical portion of enterocytes in inflamed tissue. B1R protein is significantly increased in both active UC and CD intestines compared with controls. In patients with active UC, B1R mRNA is significantly higher than B2R mRNA. However, in inactive UC patients, the B1R and B2R mRNA did not differ significantly. Thus bradykinin receptors in IBD may reflect intestinal inflammation. Increased B1R gene and protein expression in active IBD provides a structural basis of the important role of bradykinin in chronic inflammation.

The rate of tumour growth is dependent on the balance between proliferation and apoptosis at all stages of carcinogenesis. Apoptosis inhibition, in turn, depends partly on the balance between expression of two cell death regulatory genes, Bcl-2 and Bax. Colon cancer has long been associated with disturbances in apoptosis regulation. The aim of our study was to determine the expression levels of Bcl-2 and Bax mRNAs in 1 μg sample of total RNA obtained from normal colon and colon adenocarcinoma. This study was intended to evaluate possible differences in Bcl-2 and Bax mRNA levels at particular stages of colon adenocarcinoma classified according to Duke's system. The apoptotic frequency (represented by Bax mRNA copy number) was inversely proportional to the decrease of Bcl-2 gene expression. Terminal deoxynucleotidyl transferase-mediated dUTP nick-end labeling (TUNEL) was performed to confirm apoptosis.

Butyric acid, a short-chain fatty acid physiologically present in human large gut, is derived from bacterial fermentation of complex carbohydrates. It has been shown to reduce the growth and motility of colon cancer cell lines and to induce cell differentiation and apoptosis. Apoptosis is considered a result of normal colonocyte terminal differentiation in vivo. The aim of this study was to characterize the cellular mechanisms regulating differentiation of colon cancer cells stimulated with sodium butyrate (NaB). The two human colon cancer cell lines Caco-2 and HT-29 were treated with NaB at physiologically relevant concentrations. Alkaline phosphatase (ALP) activity, a marker of colonocyte differentiation, was increased 48 hr after treatment with 1 mM NaB. Higher doses of NaB (5 and 10 mM) induced apoptosis of the cells and failed to stimulate the colonocyte differentiation. Therefore, we assumed that butyrate augments cell differentiation and induces apoptosis, acting via various intracellular mechanisms, and butyrate-mediated programmed cell death cannot be considered a consequence of colonocyte terminal differentiation. The effect of NaB on ALP activity was significantly attenuated in the presence of inhibitors of protein kinase C and JNK. Inhibition of MEK-ERK signal transduction pathways augmented the impact of butyrate on colonocyte differentiation. These results suggest that butyrate could influence the colonocyte differentiation via modulation of the activity of cellular protein kinases and signal transduction.

DOPA–melanin and its complexes with netilmicin and metal ions (Zn 2+ ,Cu 2+ ) were examined by EPR spectroscopy. Broad EPR lines of o -semiquinone free radicals and paramagnetic Cu 2+ were measured. Netilmicin slightly increased free radicals concentration in DOPA–melanin. Diamagnetic Zn 2+ and paramagnetic Cu 2+ ions increased and decreased, respectively, free radical concentrations in melanin–netilmicin complexes. Higher free radical concentrations were determined for (melanin–Zn 2+ )–netilmicin complexes than for (melanin–netilmicin)–Zn 2+ . Free radical concentrations were lower in (melanin–Cu 2+ )–netilmicin complexes than in (melanin–netilmicin)–Cu 2+ . Slow and fast spin–lattice relaxation processes were found for free radicals and Cu 2+ systems in the samples. Netilmicin and Cu 2+ increased spin–lattice relaxation time in melanin.

Telomerase is a reverse transcriptase that adds repetitive telomere sequences to the end of chromosomes, which is thought to be essential for cellular immortality and oncogenesis. The enzyme consists of three subunits: human telomerase reverse transcriptase (hTERT), human telomerase RNA (hTR), and telomerase protein 1 (TP1). The hTERT subunit determines the activity of telomerase as an enzyme and is detected in most human tumors and regenerative cells. But many studies have revealed that hTR and TP1 are expressed constitutively. This resuts suggest that the hTR and TP1 subunits may be potentially good markers of endogenous RNA control. Endometrial dating was determined from the pathomorphology of the endometrium and classified into normal proliferative endometrium, endometrial hyperplasia (simple, complex, and atypical), and endometrial adenocarcinoma. The analysis of the expression of the hTERT, TP1, and hTR telomerase subunits was performed by a quantitative polymerase chain reaction method, based on fluorescent TaqMan methodology (ABI Prism 7 700 Sequence Detection System) capable of measuring fluorescence in real time. The aim of the study was an analysis of the expression profiles of genes encoding hTR and TP1 telomerase subunits in normal endometrium, endometrial hyperplasia, and adenocarcinoma forthe estimation of the possibility of once application in endogenous RNA control of gene analysis in the endometrium. The nonparametric Mann–Whitney U test and analysis of variance Friedman test were used to evaluate the variation in telomerase subunit mRNA level between normal endometrium, and endometrial hyperplasia and adenocarcinoma. The results confirm the hTR subunit expression as a good marker of endogenous control in quantitative analysis of gene transcription in endometrial tissue. TP1 subunit transcriptions have not been detected constitutively in our study.

The objective of this study was to determine the frequencies of human cytomegalovirus (HCMV) infection and HCMV genome copy number in blood of consecutive (treated from several months to several years) systemic lupus erythematosus (SLE) patients (22 women). The obtained results were compared to the healthy controls (15 women). All patients fulfilled at least four of the 1982 revised American rheumatism association (ARA) classification criteria for SLE. Our patients demonstrated three or four of the nine possible organ systems involved and most of them had mild SLE with SLE disease activity index (SLEDAI) score10 at time when blood samples were collected to detect HCMV. Quantitative analysis of HCMV genome was performed with aid of sequence analyzer ABI PRISM 7,700 Perkin Elmer. Primers and probe were constructed on the basis of IE4 region of HCMV genome. The viral load was expressed as log(10) of calculated HCMV genome copy number. Qualitative analysis revealed that 100% of our SLE patients were infected with HCMV, whereas in the control group only 73% of persons were HCMV positive. Statistically significant difference was demonstrated when the strength of the association between SLE or controls and infection of HCMV was calculated (estimated by Fisher's exact test, P value=0.02). Higher viral DNA copy number was observed in whole blood of SLE patients than in the control group (338.45+/- 221.76 and 229.00+/- 405.61 copies/ml respectively) but did not reach statistical significance level (95% confidence interval from 170.41 to 249.32, P=0.71). Furthermore percentage of patients with HCMV-DNA copy number2.0 x 10(2) copies/ml was statistically significantly higher than this one in controls. The data show association between HCMV infection and SLE, which should be taken into account during the course of SLE.

HCV virus infections have become a serious epidemiological problem throughout the world. Hepatitis C therapy includes the administration of IFN-alpha and ribavirin, but results in the complete eradication of HCV viremia in only 30% of patients. TNF-alpha is one of the factors involved in hepatitis C pathogenesis and the results of therapy. In this study, we present the results of applying real-time RT-PCR for assessing the TNF-alpha mRNA level in the peripheral blood of patients treated with IFN-alpha and ribavirin. We found the TNF-alpha mRNA level to be higher in HCV-infected patients compared with healthy controls when analyzed after 4 weeks (p = 0.001) and 3 months (p = 0.003) of IFN-alpha/RIBA therapy. The pretreatment level and the level after six months of therapy were not significantly different from the level of healthy controls. There were no significant differences in TNF-alpha mRNA levels between patients who responded to anti-HCV therapy, resulting in a decrease in HCV viremia below detection limit over 6 months and patients whose HCV RNA was not eliminated (p = 0.881). These results indicate that there is a transient increase of TNF-alpha gene expression during anti-HCV therapy. This fact may be connected with the host organism's response to IFN-alpha/RIBA therapy.

Objectives One of the important factors responsible for vessel wall remodelling is programmed cell death. In the paper the role of smooth muscle cell (SMC) apoptosis in primary varicose veins (PVV) is investigated. Material and methods Vein specimens were obtained from 40 patients with PVV. In each case proximal and distal (upper crural) great saphenous veins (GSV) were harvested. Morphometric computer assessed quantitative evaluation of SMCs, collagen and elastin content was carried out. Apoptotic cells were detected by TUNEL assay. The levels of p53 , BAX , BCLl-2 and p21 mRNA expression were assessed by real time RT-QPCR and the presence of respective proteins in the vessel wall was confirmed by immunohistochemistry. Results In the proximal GSV segments a significant increase of p53 , p21 and BCL-2 mRNA levels was found in PVV patients. In the distal segments BAX and BCL-2 expression levels were higher. Taking into account the patient age, elevated p53 mRNA expression level was noticed in the distal incompetent GSVs of young PVV patients. In this group a statistically significant increase in the apoptotic index (APIx) within the vein media was found which correlated positively with p53 mRNA expression level. There was no increase of the apoptotic activity in elderly patients that led to the structural changes increase. In proximal GSV segments, despite SMC amount reduction or presence of structural changes in perivalvular wall region, no increase of the APIx with was noticed. Conclusions P53 —related apoptosis is one of the regulatory mechanisms of vein wall homeostasis maintenance. During varicose vein development its activation is related to the early stages of the disease. In the further course, the down-regulation of the SMC apoptosis within the vein media leads to the structural changes increase. The reduction of the SMC population corresponding to an increase of p21 expression in proximal saphenous vein segments suggests that the cell cycle disturbances may lead to the ‘weakness' of the proximal GSV wall. Valve injury is not the only factor leading to the varicose veins occurrence.

Free radical properties of different types of tumor cells were compared. Electron paramagnetic resonance (EPR) studies were performed for human (BM, IGR and SK) and mouse (B16 and S91) melanoma cells. In contrast to melanotic melanoma IGR, BM and B16 cells, amelanotic S91 cells contained only a trace amount of melanin. No EPR signals were detected for Caco2 cells and only a very weak EPR line was measured for fibroblast cells. Melanin does not exist in these cells. The aim of this work was the application of EPR spectroscopy to the determination of the kind of melanin (eu- or pheomelanin) in melanotic tumor cells. Microwave saturation of EPR spectra of tumor cells with high and low melanin content was compared. Eumelanin was identified in human BM, IGR, SK, and B16 melanoma cells. Single asymmetrical EPR lines were detected for these samples. The EPR spectra of human BM melanoma cells had the highest intensity. Paramagnetic centers in amelanotic S91 melanoma cells were also found. Trace amounts of eumelanin free radicals and the other free radicals in cells were responsible for their very weak EPR lines. The obtained results indicate that EPR spectroscopy is a very useful technique for the identification of melanin in tumor cells. Strong differences of microwave saturation of EPR lines for cells with high and low melanin content were observed. EPR lines of tumor cells with a low melanin content did not saturate at the used range of microwave power. Saturation was observed for melanotic BM melanoma cells.

Changes in free radicals system of Cladosporium cladosporioides -melanin and model DOPA-melanin caused by diamagnetic Cd 2+ ions were studied by electron paramagnetic resonance (EPR) spectroscopy. EPR line of eumelanin was mainly found in the spectrum of Cl.cl. -melanin. Cd 2+ ions increased o -semiquinone free radicals concentration in both natural and synthetic melanins. Cd 2+ broadened EPR lines of Cl.cl. -melanin in mycelium and the ions fastened spin–lattice relaxation processes. The narrower EPR lines and slower spin–lattice relaxation were obtained for DOPA-melanin–Cd 2+ complexes than for DOPA-melanin. Pheomelanin additionally existing in Cl.cl. samples was responsible for differences between the EPR data for Cl.cl. melanin and DOPA-melanin.

Neuromelanin (NM) is a complex polymer pigment found in catecholaminergic neurons of the human brain. The structure, formation pathway, and physiological function of NM have not yet been clarified, but interest in this polymer has been sparked by the suggestion that NM is involved in cell death in Parkinson's disease. In the current study, pyrolysis-gas chromatography/mass spectrometry analysis was applied for structural investigation of NM isolated from the human substantia nigra, using synthetic eumelanin and pheomelanin-type pigments as reference materials. None of the heterocyclic, sulfur-containing compounds being characteristic thermal degradation products of cysteinyldopamine-derived units of synthetic pheomelanin standard was detected in the pyrolysates of natural NM. The results suggest that nigral pigment isolated from normal brain tissue does not contain benzothiazine-type monomer units. Pyrolytic experiments in the presence of a derivatizing agent allowed identification of high levels of saturated and monounsaturated straight-chain C14-C18 fatty acids and led to the conclusion that a part of a lipid component is chemically bound to the NM macromolecule. The nigral pigment was also shown to be tightly associated with an isoprenoid-type compound.

A method for the qualitative analysis of aromatic hydrocarbons in air and their various urinary metabolites is presented. The air was sampled in charcoal tubes and extracted with carbon disulfide. The hydrocarbons were identified as being aliphatic hydrocarbons (C(9)-C(19)), aromatic hydrocarbons and heterocyclic compounds. The urinary metabolites after enzymatic hydrolysis were analyzed by solid-phase extraction with a styrene-divinylbenzene resin, silylation with N,O-bis(trimethylsilyl)acetamide and GC/MS for separation and detection. Satisfactory separation of all compounds investigated was achieved without interference due to matrix peaks. The following compounds were identified in the urine of workers: dimethylphenol isomers, 4-ethyl-1,3-benzenediol, 2-ethoxybenzoic acid and methoxyphenols. Trimethylsilyl derivatives of aromatic hydroxyacids and hydroxymethoxyacids were found in the urine of occupationally exposed workers by means of a silylation procedure.

Estrogen receptor (ER)-beta may play a significant role in estrogen action in several human tissues. Estrogen receptor beta may act as a transdominant repressor of ER alpha transcriptional activity trough heterodimers form. Estrogen receptor messenger RNA (mRNA) variants also may be involved in various diseases, including endometrial cancer. The absence of estrogen receptors has often correlated with poor prognosis of endometrial tumors. The objective of the study was to determine the number of mRNA ER beta delta 6 and (wtER beta) in 1 microgram total RNA obtained from tissues of normal, hyperplastic endometrium, and endometrial adenocarcinoma. This study was designed to evaluate possible differences in the ER beta delta 6 and wtER beta messenger RNA (mRNA) level in the normal, hyperplastic endometrium, and endometrial endometrioid adenocarcinoma (G1, G2 morphological degree). Adenocarcinoma showed significantly lower ER beta delta 6 mRNA level than proliferative (p = 0.032) and secretory (p = 0.01) endometrium. A decrease of mRNA wtER beta in endometrial adenocarcinoma (p = 0.006) also was observed.

Properties of butyltin complexes [Sn(C4H9-n)3{OOCC6H3(NH2)2-3,4}]n (1), [Sn(C4H9-n)3{OOCC6H3(NH2)2-3,5}] (2), [Sn(C4H9-n)3{OOCC6H4NNC6H4N(CH3)2-4}] (3) and [Sn(C6H5)3{OOCC6H3(NH2)2-3,5}]n (4) have been investigated. 1H, 13C and 119Sn NMR spectra indicate that the compounds in chloroform are distorted tetrahedral and in strongly coordinating solvents trigonal-bipyramidal complexes. Structure of complex 3 has been determined by X-ray crystallography. This compound adopts trigonal bipyramidal structure with bridging carboxylato ligand bound asymmetrically with tin atoms in axial positions. The complexes are effective cytostatic agents.

Peroxynitrite—a strong oxidizing and nitrating agent—is thought to be a mediator of the cytotoxic action of nitric oxide in biological systems. There is some evidence that peroxynitrite-dependent processes may be involved in the pathomechanism of Parkinson's disease. Recently, it has been suggested that neuromelanin, present in the cytoplasm of nigrostriatal neurons, can act as natural scavenger of peroxynitrite. In this study the effect of peroxynitrite on the structure of synthetic models of human neuromelanin was investigated. Model neuromelanins, obtained from dopamine (DA) and/or 5- S -cysteinyldopamine (5- S -CysDA), were pyrolyzed at 770 °C before and after treatment with peroxynitrite and the thermal degradation products separated by gas chromatography were identified by mass spectrometry. After exposure to peroxynitrite, reduced content of the typical pyrolysis products accompanied by elevated levels of low molecular weight gases in the pyrolysates were observed. The changes in pyrolytic patterns were dependent on melanin type and peroxynitrite concentration. It was concluded that interaction with peroxynitrite causes extensive oxidative degradation of the analyzed pigments. This may be of great biological importance with regard to depigmentation of nigrostriatal neurons demonstrated in Parkinson's disease.

Mutations in sodium channel alpha-subunit gene (SCN5A) result in multiple arrhythmic syndromes, including long QT3 (LQT3), Brugada syndrome (BS), an inherited cardiac conduction defect, sudden unexpected nocturnal death syndrome (SUNDS) and sudden infant death syndrome (SIDS), constituting a spectrum of disease entities termed Na+ channelopathies. These diseases are allelic disorders, if not the same disease with variable penetrance and variable modifiers worldwide. Interestingly, death occurs during sleep in all of these disorders, suggesting a common mechanism. To date, mutational analyses have revealed about 103 distinct mutations in SCN5A, of which at least more than 30 mutations are associated with LQT3, whereas the rest of the mutations are affiliated with the remaining sodium channel disorders. The majority of these mutations are missense. However, other types such as deletions, insertions, frameshifts, nonsense and splice-donor errors have also been reported.

Electron paramagnetic resonance (EPR) spectroscopy was used to examine free radical properties of model neuromelanins obtained by oxidative polymerization of noradrenaline, adrenaline and dopamine. We compared the lineshape of the experimental spectra, type and concentration of free radicals in the analyzed samples. The effect of different concentrations of Cu2+ on free radicals in melanins was studied. The total concentration of free radicals (about 1018 to 1019 spin/g) in the studied melanins increases as follows: adrenaline-melanin < dopamine-melanin < noradrenaline-melanin. EPR spectra of dopamine-melanin and adrenaline-melanin were a single EPR line (ΔB pp, 0.50 and 0.55 mT, respectively). o-Semiquinone free radicals with the characteristicg-value of 2.0040 exist in these melanins. EPR spectra of noradrenaline-melanin were a superposition of two lines (ΔB pp, 0.45 and 0.81 mT). o-Semiquinone free radicals were responsible for the narrower component. Nitrogen free radicals with ag-factor of 2.0030 were probably responsible for the broader component. Paramagnetic copper ions quenched the EPR signals of melanin free radicals in the studied samples. For melanin-Cu2+ complexes, broad EPR lines (ΔB pp, 10–32 mT) of copper ions with ag-value of about 2.1 appeared. The influence of the microwave power on the EPR spectra of these complexes demonstrated the fast spin-lattice relaxation in the copper system in melanins.

Effect of metal ions on free radical properties of natural melanin produced by soil fungiCladosporium cladosporioides was studied. The electron paramagnetic resonance (EPR) spectrum of the studied melanin consists mainly of a single line of eumelanin, and only a very weak signal of pheomelanin was observed. o-Semiquinone free radicals form paramagnetic centers in melanin. Diamagnetic Zn2+ ions produce an increase in the free radical concentration in melanin. Quenching of melanin EPR lines was obtained for melanin and paramagnetic Cu2+ ion complexes. Slow spin-lattice relaxation processes are characteristic for the free radicals in melanin samples and fast spin-lattice relaxation was observed for Cu2+ ions. The EPR lines of copper ions saturate at higher microwave powers than the EPR lines of melanin.

The profile of tissue kallikrein (TK) and its inhibitor, kallistatin was evaluated in patients with active ulcerative colitis (UC) and Crohn's disease (CD). Tissue kallikrein is mainly localized to goblet cells and kallistatin to epithelial cells of human intestine. Intestinal tissue kallikrein (ITK) and kallistatin are significantly decreased in inflamed intestine compared to noninflammatory controls. TK mRNA as well as kallistatin mRNA is significantly decreased in intestinal biopsy samples from UC-active patients compared with controls. The difference in distribution and levels of ITK and kallistatin in protein and mRNA in patients with inflammatory bowel disease (IBD) compared to controls suggest a role in inflammatory state.

Electron paramagnetic resonance (EPR) studies of complexes of chloroquine with melanin synthesized from 3,4-dihydroxyphenylalanine (DOPA) confirmed an important role ofo-semiquinone free radicals during the drug binding by melanin. Chloroquine increases free radical concentration in DOPA-melanin. It has been also demonstrated that chloroquine binding to melanin is modified by metal ions. Co2+ ions reduce and Zn2+ ions increase the concentration of free radicals in both DOPA-melanin and melanin-chloroquine complexes. Free radical properties of melanin strongly depend on the presence of oxygen (O2) in the environment of the sample. Interactions of melanin and oxygen molecules cause a decrease of the free radical concentration in the samples and a narrowing of the EPR lines. Higher values of both spin-spin and spin-lattice relaxation times were determined for melanin samples in air than for evacuated samples. Application of the observed effects in oximetry is suggested.

Summary Arbitrarily primed polymerase chain reaction (AP-PCR) method was applied to the differentiation of 15 (soil and intestinal) Desulfovibrio desulfuricans strains. The primer M 13, which is a core sequence of phage M 13, was found to be appropriate for the differentiation of isolates of this species. The analysis revealed characteristic band patterns for all of the examined strains of which two soil strains (DV-7 and DV-8) showed identical DNA fingerprints. According to Jaccard’s coefficient, the soil bacterial group as well as intestinal bacterial group formed two different clusters. Furthermore, the soil strains showed greater variability than the intestinal isolates. Based on the AP-PCR fingerprints D. desulfuricans strains were differentiated depending on their origin. This study demonstrates that the typing method AP-PCR can be useful in epidemiologic investigations as a rapid and valuable tool for differentiation of the strains of D. desulfuricans species.

The properties of vinyltin and phenyltin complexes [Sn(CH=CH2)3{μ-OOCC6H3(NH2)2-3,4}]n (1), [Sn(C6H5)3{OOCC6H3(NH2)2-3,4}] (2), [Sn(C6H5)3{OOC-2-C6H4N=NC6H4N(CH3)2-4}] (3) and [Sn(CH=CH2)3{OOC-2-C6H4N=NC6H4N(CH3)2-4}] (4) have been investigated. The structures of complexes 1, 2, and 3, have been determined by X-ray crystallography. Compound 1 is a distorted trigonal-bipyramidal complex and compounds 2 and 3 adopt a distorted tetrahedral structure. Complex 1 is a single-strand polymer with a bridging 3,4-diaminobenzoato ligand coordinating via the O(1) atom of the carboxylato group and the nitrogen atom of the para-amino group. The oxygen and nitrogen atoms occupy the axial coordination sites. The Sn(1)−N(2A) bond is weak. In complexes 2 and 3 the carboxylato ligands are strongly coordinated to the central atom via one oxygen atom, and the Sn(1)−O(2) distances are considerably longer. Weak interactions of the central atom with the amino group in complex 1, and with the O(2) atoms in complexes 2 and 3, as well as the hydrogen bonds, stabilize the crystal structure. The complexes are effective cytostatic agents. (© Wiley-VCH Verlag GmbH, 69451 Weinheim, Germany, 2002)

The structure of the tin(III) complex [Sn 2 (CH 2 CH 2 CN) 6 ] has been determined. There are two independent molecules in the crystal, both adopt distorted eclipsed conformation. The molecular and electronic structures of this compound have been studied both at the semiempirical level and with the use of non-empirical ab initio methods. The calculated Sn-Sn distances agree well with those found crystallographically. The results of calculations showed that the eclipsed conformation of complex is more stable as compared with staggered conformation. The compound show modest cytotoxic activity against A549 and HSMC cells. All rights reserved.

Synthetic neuromelanins obtained from dopamine and/or 5- S -cysteinyldopamine were pyrolyzed at 770 °C. The thermal degradation products were separated by gas chromatography and identified by mass spectrometry. Pyrolytic patterns of melanins containing 5- S -cysteinyldopamine-derived component were different from that of dopamine-melanin. Major pyrolytic products of 5- S -cysteinyldopamine-originated units were indentified as 2H-1,4-benzothiazin-5-one, 4-hydroxybenzothiazole and thiazoloisoquinoline and their alkyl derivatives. The results obtained indicate that pyrolysis-gas chromatography-mass spectrometry method is suitable for identification of 5- S -cysteinyldopamine-derived units in synthetic melanin copolymers and it could be applied for structural analysis of natural neuromelanin.

The properties and structures of the tetranuclear dibutyltin complexes [Sn 4 (μ 3 -O) 2 (C 4 H 9 -n) 8 {OOCC 6 H 3 (NH 2 ) 2 -3,4) 4 ] (1), [Sn 4 (μ 3 -O) 2 (C 4 H 9 -n) 8 {OOCC 6 H 3 (NH 2 ) 2 -3,5} 4 ] (2), [Sn 4 (μ 3 -O) 2 (C 4 H 9 -n) 8 {OOC-2-C 6 H 4 N=NC 6 H 4 N(CH 3 ) 2 -4} 4 ] (3) are described. Complex 3 adopts a structure with a tetranuclear Sn 4 (μ 3 -O) 2 core. All tin atoms are five-coordinate and form bonds with three oxygen atoms and two butyl ligands. Two carboxylates are bridging and two are terminal ligands. IR and NMR spectra indicate that the same structure is adopted by complexes 1 and 2. The molecular and electronic structures of complex 1 of C i symmetry have been studied using the semi-empirical PM3 formalism. The calculated structure and bond distances agree with X-ray data. All complexes are effective antitumor agents.

Peroxynitrite-mediated linoleic acid oxidation and tyrosine nitration were analysed in the presence of synthetic model neuromelanins: dopamine (DA) -melanin, cysteinyldopamine (CysDA) -melanin and various DA/CysDA copolymers. The presence of melanin significantly decreased the amount of 3-nitrotyrosine formed. This inhibitory effect depended on the type and concentration of melanin polymer. It was found that incorporation of CysDA-derived units into melanin attenuated its protective effect on tyrosine nitration induced by peroxynitrite. In the presence of bicarbonate, the melanins also inhibited 3-nitrotyrosine formation in a concentration dependent manner, although the extent of inhibition was lower than in the absence of bicarbonate. The tested melanins inhibited peroxynitrite-induced formation of linoleic acid hydroperoxides, both in the absence and in the presence of bicarbonate. In the presence of bicarbonate, among the oxidation products appeared 4-hydroxynonenal (HNE). CysDA-melanin inhibited the formation of HNE, while DA-melanin did not affect the aldehyde level. The results of the presented study suggest that neuromelanin can act as a natural scavenger of peroxynitrite.

The effects of dopamine-melanin (DA-melanin), a synthetic model of neuromelanin, on peroxynitrite-mediated 3-nitrotyrosine formation, oxidation of tryptophan in bovine serum albumin and inactivation of erythrocyte membrane Ca(2+)-ATPase activity were investigated in the absence and in the presence of bicarbonate. DA-melanin inhibited nitration of free tyrosine, loss of tryptophan residues and Ca(2+)-ATPase inactivation by peroxynitrite in a dose dependent manner. In the presence of bicarbonate, this inhibitory effect was lower for nitration and insignificant for oxidative protein modifications. These results suggest that neuromelanin can protect against nitrating and oxidizing action of peroxynitrite but is a worse protector against the peroxynitrite-CO(2) adduct. As peroxynitrite may be a mediator of neurotoxic processes, the obtained results suggest that neuromelanin may be important as a physiological protector against peroxynitrite.

Properties of melanins depend strongly on their chemical constitution. Melanins from differently colored strains of Drosophila melanogaster demonstrate various proprieties, and biological activity in the presence of toxic ions or radiation. In the presented work, we analyzed chemical constitution of the melanins derived from black, gray and yellow D. melanogaster strains. Analysis of the products forming during thermal degradation of the biopolymers by pyrolitic gas chromatography followed by mass spectrometry allowed us to determine chemical composition of these biopolymers and classified melanins derived from black and gray strains as eumelanins and derived from yellow strains as pheomelanins. These findings allow us to determine chemical and biological properties of the melanins in vivo, and can explain susceptibility of differently colored strains of D. melanogaster to metal ions.

The effect of dopamine-melanin (DA-melanin), synthetic model of neuromelanin, on the stability of 13-hydroperoxyoctadecadienoic acid (13-HPODE) has been investigated using a reverse-phase high-performance liquid chromatography (HPLC) with UV detection. It was found that DA-melanin effectively accelerated the decomposition of 13-HPODE, both in the absence and in the presence of ferrous ions. The disappearance of 13-HPODE was accompanied by the formation of 13-hydroxyoctadecadienoic acid (13-HODE). The results obtained indicate that DA-melanin is able to reduce linoleic acid hydroperoxide to its stable hydroxyl derivative. The fatty acid hydroperoxide-reducing ability of DA-melanin appears to play an important role in the antioxidative activity of neuromelanin in the process of lipid peroxidation.

Dimeric rhodium(II) acetato (OAc), lactato, and mandelato (mand) complexes with phenanthrolines [Rh 2 (OOCR) 2 (NN) 2 (H 2 O](OOCR) 2 (R = CH 3 , CH 3 CHOH, C 6 H 5 CHOH, NN = 1,10-phenanthroline (phen) and 4,7-diphenyl-1,10-phenanthroline (dpp)) have been synthesized and characterized using electronic, IR, and NMR spectra. Cytostatic activity of these complexes against the synchronously cultivated green alga Chlorella vulgaris has been investigated. The obtained results indicate that cationic complexes [Rh 2 (RCOO) 2 (NN) 2 (H 2 O) 2 ] 2+ show higher cytostatic activity than [Rh 2 (OAc) 4 (H 2 O) 2 ].

The content of endogenous melanin in white Balb c and black C-57 Bl. mice affected Zn2+ and Co2+ accumulation, elimination and distribution following intraperitoneal administration of these ions. Elimination of Co2+ ions was significantly greater in white mice than in black mice. Accumulation of Zn2+ was higher than that of Co2+ and did not depend on melanin content in mice. The same response was observed upon administration of exogenous melanin. Both ions accumulated mainly in spleen and heart irrespective of melanin content.

The inhibiting effect of melanin synthesized from dihydroxyphenylalanine (DOPA), dopamine, adrenaline and adrenolutin on the ultraviolet- or the Fe(2+)-ascorbic acid-induced peroxidation of cardiolipin liposomes has been studied. All these melanins are able to inhibit both the ultraviolet- and the Fe(2+)-ascorbic acid-induced lipid peroxidation. Antioxidative activity of melanins enhances in the order: dopamine-melanin less than melanin synthesized from dopamine in the presence of Cu(2+) less than DOPA--melanin less than melanin synthesized from adrenaline in the presence of Cu(2+) approximately equal to adrenolutin-melanin, and correlates with their ability to scavenge superoxide anion radical. The optical screening effect of the investigated melanins in the inhibition of lipid peroxidation was not higher than 15% for the most active melanins.

Cardiovascular diseases including myocardial infarction cause 50 percent of all deaths cases and range before cancer as the cause of death. The term 'ischemic heart disease' covers wide range of diseases including all ischemic conditions of myocardium. It's the most common reason (98%) is atherosclerosis, which causes a restriction or occlusion of a coronary artery lumen resulting in myocardial ischemia. Acute coronary syndromes are caused by the rupture of unstable atherosclerotic plaque resulting in clot formation which blocks the blood vessel. In this process an important role play adhesion molecules triggering adhesion, aggregation and the whole blood coagulation cascade.The aim of the study was to assess PECAM-1 gene expression in peripheral blood mononuclear cells in patients with acute coronary syndrome during first 24 hours of hospitalization in comparison with healthy individuals.There were 80 subjects included to the study divided into two groups. First group, consecutive patients admitted to the Department of Cardiology due to acute coronary syndrome and the second group, 20 healthy subjects. The total RNA was extracted from peripheral blood mononuclear cell (PBMC) using Chomczynski and Scchaci method. Transcription activity was assessed using commercially available TaqMan Gene Expression Assays. The PECAM-1 gene PCR reaction was preformed with ABI PRISM 7000 Sequence Detector (Applied Biosystems, USA).The comparison of PECAM-1 gene expression revealed statistically significant difference, between increased level of PECAM-1 in patients with acute coronary syndrome and it's lower level in healthy individuals.Observed in peripherial blood mononuclear cells increase of PECAM-1 protein gene expression may be responsible for increased adhesion and aggregation process and acute coronary syndrome occurrence.

Cardiac syndrome X is typically characterized by effort induced anginal pain with ST segment depression suggestive of myocardial ischemia and normal coronary arteries at angiography. The possible mechanism that may participate in the pathology of CSX is a microvascular dysfunction related to inflammatory process affecting endothelium. Interferon gamma (IFN-gamma) is an important cytokine in inflammatory reaction. It acts through its specific receptor composed of 2 subunits IFN-gamma R1 (ligand binding) and R2 (signal transduction). The expression and proportion of these subunits influences IFN-gamma activity. The aim of the study was to assess the gene expression of IFN-gamma and its receptors in peripheral blood mononuclear cells (PBMC) from patients with syndrome X.The study was carried out in 36 patients aged 44-77 (average 57 years old) with cardiac syndrome X and 23 sex- and age-matched healthy subjects (control group). To evaluate gene expression of IFNgamma and its receptor total mRNA was extracted from peripheral blood mononuclear cells (PBMC) and the number of mRNA copies were assessed by quantitive reverse transcriptase polymerase chain reaction (QRT-PCR).We have not observed statistically significant differences in INFgamma gene expression between studied group and control. Genes encoding IFNgamma receptor subunits showed higher expression in PBMCs from patients with cardiac syndrome X vs control subjects (IFNgammaR1, 97,244 +/- 26,956 c/microg vs 12,120 +/- 2,940 c/microg, p0.005, respectively and IFNgammaR2, 129,153 +/- 36,883 c/microg vs 16,445 +/- 2,923 c/microg, p0.005, respectively).Variation in transcriptional activity of genes encoding INF-gamma receptor subunits may affect function of microvasculature and thereby participate in the pathology of cardiac syndrome X.

The cytomegalovirus infection is most common causes of intrauterine infection of the fetus. Using of serologic diagnostic methods the kind of infection is unknown. The aim of the study was assessment of the risk of CMV infection depending of genome account in mother's blood and I amniotic fluid. The study was performed in choosen pregnancies, in which we expected cytomegalovirus infection using serological criteria. In prenatal diagnostic CMV infection using QPCR, the best material is amniotic fluid. Mother's blood assessment of CMV genome count does not make growth diagnostic possibility of the assesement of transmission the infection from mother to fetus.

Anorexia nervosa is a serious eating disorder with the highest mortality rate of any psychiatric disorder. The DSM-IV classification differentiates two AN types: the restricting type (AN-R) and the binge-eating/purging type (AN-BP). Leptin (LEP) levels can be thought of as a signal to the body of its energy reserves. The leptin receptor (including all its mRNA isoforms) is expressed in many tissues. Our aim was to discover the transcript expression profile of the LEP receptor-coding gene in the peripheral blood mononuclears in AN-R and AN-BP patients. Three young women suffering from Anorexia nervosa (one with AN-BP and two with AN-R) took part in the study, along with three non-anorexic subjects as our reference group. LEP receptor gene expression was examined using the oligonucleotide microarray method (HG-U133A, Affymetrix). The results were normalized using RMAExpress. Next, the accumulation analysis method was used (clustering). Hierarchical clustering resulted in three groups of separate clusters. The first group (cluster I) consisted of AN-R patients. The next group (cluster II) consisted of reference group patients suffering from different psychic disorders not related to eating disorders. Cluster III consisted of two patients — the first with AN-BP and the second with an adaptive disorder.

Toxoplasma gondii infection during pregnancy is still a difficult problem in the contemporary perinatology. Difficulties met during interpretation of serological tests carried out in pregnant patients to detect Toxoplasmosis implies more and more frequent use of the Polymerase Chain Reaction (PCR).To evaluate the dependence between serological tests and quantity of the Toxoplasma gondii genomes in mothers' blood and amniotic fluid or neonatal blood, the quantitative PCR (q-PCR) method was applied.The analysis was performed in 81 pregnant women. Maternal blood, amniotic fluid and newborns' umbilical blood samples were evaluated for the presence of Toxoplasma gondii DNA. IgG and IgM Toxoplasma gondii antibodies were evaluated by the ELISA method.High seroprevalence (51.9%) of the Toxoplasma gondii was confirmed. Toxoplasma gondii genetic material in blood and/or amniotic fluid was found in 33 patients. It was stated that quantity of the protozoa and anti-IgM presence in mothers' blood are the factors influencing significantly the Toxoplasma gondii manifestation in amniotic fluid.High suitability of PCR in diagnosis of Toxoplasmosis during pregnancy and vertical transmission was confirmed.

Domestic pig may serve as the most appropriate organ source for human xenotransplantation in the future. However, there is a serious threat of xenogeneic pathogens transmission, especially porcine endogenous retroviruses (PERVs) which are present in genomes of all pigs. The aim of this study was to monitor the prevalence and distribution of PERV DNA in organs of a domestic pig.We used a primer set for a highly conserved fragment of PERV gag sequence to monitor a total PERV DNA copy number and genotype-specific primer sets to study PERV subtypes distribution using Real-Time QPCR (SYBR Green I).Our results showed that PERV DNA was present in all studied pigs, however, most PERV DNA molecules carried numerous mutations thus indicating inability to express functional retroviral particles. The level of PERV DNA in kidney was much higher than in heart (p = 0.007) and in the liver (p = 0.009).It indicates that kidney is potentially the biggest PERV reservoir which makes it the organ of particular concern in xenotransplantation. We also conclude it is possible to monitor pig herds for individuals with the lowest PERV DNA prevalence, especially lacking PERV-C, and perhaps with only defective PERV proviruses that are unable to express functional RNA.

Hypertrophic cardiomyopathy (HCM) is a genetic-based disease. Several gene mutations leading to HCM development have been described.Detailed examination of phenotype and genotype of a family with HCM.Clinical and genetic examinations were performed in a family with HCM, in which 3 sick persons with different disease phenotype were found.In all sick persons the same molecular substitution G-A (AGG-AAG) was noticed. It led to substitution Arg780-Lys in exon 21 beta-myosin heavy chain gene, which was responsible for the development of the disease. Insertion- deletion polymorphism analysis in ACE gene revealed D/D (deletion/deletion) genotype in proband and D/I (deletion/ insertion) phenotype in his mother and sister, who were heterozygous. Polymorphism A1166C analysis in AT1 gene revealed the presence of genotype A/A in proband and A/C in his mother and sister. In proband and his sister a very similar phenotype was observed, whereas they had different polymorphism for ACE gene and angiotensin 1 receptor gene. In sick proband's mother, who had phenotype different to her children, the same polymorphism as in his daughter was noticed.In the described family with HCM, different phenotype and polymorphism of ACE and AT1 genes were found.

Recent data report altered gene expression of numerous pro- and anti-inflammatory factors involved in pathology of acute coronary syndromes (ACS). Transforming growth factor beta (TGFbeta) signaling is engaged in a wide range of processes. Its effect on vessels seems to be protective due to its anti-inflammatory and anti-atherogenic action. However, it also seems to be engaged in such negative effects as neointima formation and fibrosis. The aim of the study was to assess the expression of the genes encoding TGFbeta and its receptors (type I, II, and III) in patients with ACS.The study was carried out on 24 patients with acute coronary syndrome (7 with unstable angina [UA] and 17 with myocardial infarction [MI]) and 10 age-matched healthy subjects (control). To evaluate gene expression of TGFbeta and its receptors total mRNA was extracted from peripheral blood mononuclear cells (PBMC) and the number of mRNA copies were assessed by quantitative reverse transcriptase polymerase chain reaction (QRT-PCR).MI and UA patients demonstrated significantly lower TGFbeta gene expression compared to control (2789+/-418 c/microg vs. 20262+/-2548 c/microg; p0.001, and 3390+/-518 c/microg vs. 20262+/-2548 c/microg; p0.001, respectively), as well as noticeably lower transcriptional activity of genes encoding its type I (3295+/-447 c/microg vs. 12859+/-1929 c/microg; p0.001, and 3258+/-721 c/microg vs. 12859+/-1929 c/microg; p0.01, respectively) and type II receptors (2364+/-346 c/microg vs. 19003+/-2357 c/microg; p0.001, and 2680+/-522 c/microg vs. 19003+/-2357 c/microg; p0.01, respectively). Also, gene expression of the type III receptor was inferior in the studied group compared to the control, although the difference was significant only for the UA group vs. control. Expressions of the studied genes did not differ between patients with MI and those with UA.Our report shows that the decreased activity of TGFbeta in patients with ACS is at least partly due altered transcriptional activity of genes encoding both TGFbeta and its receptors, what may be responsible for the evolution of atherosclerotic lesions.

Sulphasalazine (salicyl-azo-sulphapiridine, SAS) is a drug commonly used in the treatment of non-specific inflammatory bowel diseases, such as ulcerative colitis and Crohn disease. Chronic inflammatory states of the colon can lead to neoplastic changes of the intestinal mucosa. There are some suggestions in the literature that the intestinal bacterial azo-reductases are involved in biotransformation of SAS into 5-aminosalicylic acid (5-ASA) and sulphapyridine (SP). For this reason, it seemed worth of investigating whether transformation of SAS could be performed by the colon epithelial cells themselves. No enzymatic systems presumably exist in Caco-2, which could be responsible for SAS metabolism, because after 72 h-incubation of cell cultures with 1 mM SAS, its metabolites i.e., 5-ASA and SP were not detected in cells, neither in culture media. SAS metabolism, therefore, seems to depend on the presence of intestinal bacterial enzymatic systems. It was confirmed that 5-ASA is converted by Caco-2 cells to N-acetyl-5-aminosalicylic acid (Ac-5-ASA), which migrates to the culture medium. The other metabolite of SAS i.e., SP, was not transformed in the human colon cancer cells at all.