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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.

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.

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.

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.

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.

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.

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.

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 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 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 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.

Structural differences in melanins were studied by Curie-point pyrolysis-gas chromatography-mass spectrometry, electron spin resonance spectroscopy and chloroquine binding. Pyrolysis-gas chromatographic data on the polymer allow the calculation of a UU index that expresses the relative amount of unindolized DOPA-derived units in the melanins. It is shown that the content of unindolized units in the polymer depends strongly on the oxidation time and DOPA concentration used during the melanin synthesis. Comparison of the chloroquine binding to melanins and the pyrolysis data showed that there is an inverse relationship between the affinity of chloroquine and the UU index.

Melanins synthesized from adrenaline and dopamine in the presence or absence of copper ions were characterized by pyrolysis-gas chromatography-mass spectrometry and by IR and ESR methods. It was shown that Cu2+ are able to induce changes in the melanin structure. Melanins obtained from adrenaline-Cu2+ and dopamine-Cu2+ complexes are composed mainly from monomeric units of the indole type. Melanins synthesized from these catecholamines without Cu2+ contain additionally large amounts of unindolized monomeric units. The structure differences in both types of melanins are reflected in their sorptive abilities and spectroscopic characteristics.

The infrared and electron spin resonance spectra of synthetic 3,4-dihydroxyphenylalanine (DOPA) and tyrosine melanins and chemically modified melanin samples were determined, and it was shown that unmodified and reduced DOPA melanins exhibited similar ir spectra. Oxidized DOPA melanins showed a higher number of carboxy groups in the sample. A significant increase of free radical content in reduced DOPA melanin and a decrease of free radical content in oxidized DOPA melanin in comparison to unmodified samples were demonstrated by the use of ESR methodology. Methylation of tyrosine melanin with an excess of diazomethane gave very rich ir spectra as compared to melanins methylated with methanol saturated by gaseous HCl. In tyrosine melanin samples the esterification of carboxy groups with methanol caused a decrease in the free radical content. When diazomethane was used, the methylated melanin samples had free radical levels reduced to only about 4% of the total observed for unmodified tyrosine melanin.

Electron spin resonance (ESR) studies of chloroquine complexes with synthetic DOPA-melanin, synthetic catechol-melanin, melanin isolated from bananas and humic acid were performed. Two chloroquine concentrations (5 X 10(-5) M and 1 X 10(-3) M) were used for chloroquine-melanin complex formation. ESR studies showed differences in free radicals concentration depending on melanin origin. It was demonstrated that addition of chloroquine to melanin results in broadening of the melanin ESR signal and is accompanied by changes in the integrated intensity of the analyzed melanin samples.

The non-rotational hydration of DNA and its correlation time have been investigated by the NMR spin-echo technique. Relaxation times T 1 and T 2 were measured at 20° and the dependence of the values of T 1 and T 2 on the DNA concentration was established. The theory of this phenomenon is discussed. The non-rotational amount of water was shown to be dependent on the molecular weight of the DNA, ranging from about 8 to 10% with molecular weights of 6·10 6 to 7·7·10 6 . The non-rotational hydration represents only a part of the total water present in hydration shells. Denaturation of the DNA decreases the non-rotational hydration 1.45 times as compared with native DNA simultaneously resulting in an increase in the correlation time, τ c , by a factor of 1.45. Denatured DNA molecules have distinctly less freedom of movement than double-helix DNA which has a high degree of structural organization.

The longitudinal T1 and transversal T2 magnetic relaxation times of DNA solutions were measured at various ionic strength at elevated and lowered temperatures. From the dependence of T1 and T2 values on temperature the non-rotational hydration of DNA molecules in the native and denatured state and also during helix-coil transition was calculated. Dehydration of native DNA molecules was observed at elevated temperature. A higher ionic strength of DNA solutions is favourable for stronger dehydration at low temperatures. At temperatures close to the Tm value, determined spectrophotometrically at 260 nm (73.5, 89.5 and 99° for DNA at 0.02, 0.15 and 1 M NaCl, respectively), the DNA molecules seem to be completely dehydrated. At lowered temperatures rehydration of denatured DNA molecules was observed. However, at each temperature the hydration of denatured DNA has an average value of only 42 % of that observed for native DNA.