Expression of genes encoding kinin receptors in peripheral blood mononuclear cells from patients with acute coronary syndromes.

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 < 0.01; UA vs C: 2.13 +/- 0.98 vs 0.36 +/- 0.04; P < 0.05 respectively). B2R gene mRNA level was markedly lower in MI group versus C group (24 216 +/- 5409 copies/microg vs 39 908 +/- 5309 copies/microg; P < 0.05). The difference in B1R gene expression between MI and C group was negligible. We have not observed differences in studied genes expression between UA and C groups. CONCLUSION: Patients with ACS show inverted B1R/B2R ratio. Such disturbance in kinin signalling may reflect increased activation of circulating mononuclears, which are important participants of atherosclerotic plaque development and eventually rupture.

Differential diagnosis of gingival hyperplasia based on IFN-gamma-stimulated gene expression using RT-PCR.

Epulus is a benign gingival tumour of unknown aetiopathogenesis. Classification is inconsistent, and standard management strategies are lacking. Epuli are generally believed to be inflammatory rather than neoplastic lesions. The literature does not present any molecular analysis of the tumour characteristics. The purpose of the present study was to compare benign (epulus) and malignant (cancer) gingival hyperplasias with regard to the activity of the genes of apoptosis, proliferation, and inflammation using RT-PCR. The investigation involved 70 patients with epuli and 15 patients with gingival squamous cell carcinoma. Each subject had specimens collected from the tumour, tissue margin (incision line), and healthy tissue. Molecular investigations by RT-PCR were used to evaluate expression levels of the genes associated with apoptosis (Bcl-2, Bax, Bcl-2/Bax), proliferation (H3 histone), and inflammatory processes (IFN-gamma, IFNgamma-R1, IFN-gammaR2, IFN-gammaR1/IFN-gammaR2). Correlations have been disclosed between apoptosis and proliferation genes expression in giant cell epuli and high-differentiated gingival squamous cell carcinoma. In RT-PCR molecular analysis, giant cell epulus shows characteristics of a neoplastic lesion, while other epulus types seem to be inflammatory tumours.

Preliminary report of expression of bax in oral cavity pathologies.

Bax is considered one of major effectors of apoptosis--programmed cell death. Immunohistochemical analysis of in vitro patterns of bax expression was mostly investigated in mammalian cell lines and tissues. The present study is the first in vivo molecular analysis of bax expression in oral cavity pathologies. The study population consisted of 45 patients with hyperplasia, neoplasm in situ malignancy, and carcinoma. Biopsies were taken from incision line, tumour section, and healthy tissue. bax expression was investigated depending on the site of biopsy material sampling and final histopathology result. No statistically significant difference was demonstrated in bax expression between four hyperplasia subgroups. However, statistically significant differences in bax expression were found between the three basic study groups (P = 0.001). Statistically significant differences in bax expression were demonstrated depending on tissue collection site (P = 0.0002). We conclude that differences in bax expression may play a role in the pathogenesis of neoplastic disease.

Antioxidative activity of synthetic melanins. Cardiolipin liposome model.

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.

Catecholamine melanins. Structural changes induced by copper ions.

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.

Dopamine-melanin protects against tyrosine nitration, tryptophan oxidation and Ca(2+)-ATPase inactivation induced by 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.

Sequence analysis of proviral DNA of porcine endogenous retroviruses.

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.

Studies of the mechanism of chloroquine binding to synthetic DOPA-melanin.

In order to elucidate the mechanism of drugs binding to melanin, effects of pH, ionic strength and organic solvent on the interaction of chloroquine with synthetic dopa-melanin were studied. The results indicate that electrostatic, hydrophobic and van der Waals' forces participate in the formation of the chloroquine-melanin complex. Binding analysis by the Scatchard method showed that two classes of binding sites take part in the complex formation: strong binding sites with the association constant k1 approximately to 10(5) and weak binding sites with K2 approximately 10(4). Experiments with chemically modified melanin yielded some information about binding sites of this biopolymer. The obtained results suggest that strong binding involves both hydrophobic interaction and electrostatic attraction between the protonated ring system of chloroquine and the ortho-semiquinone groups of melanin. However, the weakly reacting sites can be identified as ionic bonds between protonated aliphatic nitrogen of chloroquine molecule and carboxyl groups of melanin. Van der Waals' forces occurring at the conjunctions of the aromatic rings of the drug and the aromatic indole-nuclei of the melanin probably take part in the weak binding too.

Electron spin resonance studies of chloroquine-melanin complexes.

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 relationship between microbial metabolic activity and biocorrosion of carbon steel.

The effect of metabolic activity (expressed by generation time, rate of H2S production and the activity of hydrogenase and adenosine phosphosulphate (APS)-reductase enzymes) of the 8 wild strains of Desulfovibrio desulfuricans and of their resistance to metal ions (Hg2+, Cu2+, Mn2+, Zn2+, Ni2+, Cr3+) on the rate of corrosion of carbon steel was studied. The medium containing lactate as the carbon source and sulphate as the electron acceptor was used for bacterial metabolic activity examination and in corrosive assays. Bacterial growth inhibition by metal ions was investigated in the sulphate-free medium. The rate of H2S production was approximately directly proportional to the specific activities of the investigated enzymes. These activities were inversely proportional to the generation time. The rate of microbiologically induced corrosion (MIC) of carbon steel was directly proportional to bacterial resistance to metal ions (correlation coefficient r = 0.95). The correlation between the MIC rate and the activity of enzymes tested, although weaker, was also observed (r = 0.41 for APS-reductase; r = 0.69 for hydrogenase; critical value rc = 0.30, p = 0.05, n = 40).

The effect of endo- and exogenous melanin on Zn2+ and Co2+ elimination and distribution in mice.

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.

Peroxynitrite mediated linoleic acid oxidation and tyrosine nitration in the presence of synthetic neuromelanins.

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.

Interaction of methotrexate with melanins and melanosomes from B16 melanoma.

It has been demonstrated that methotrexate forms stable complexes with melanin and melanosomes isolated from B16 melanoma. The number of binding sites and binding constants for methotrexate binding by intact melanosomes and melanin were n = 0.046 mumol/mg, K = 0.32 x 10(4) M-1 and n = 0.063 mumol/mg, K = 1.08 x 10(4) M-1, respectively. Binding of methotrexate to synthetic DOPA-melanin used for comparison also shows a single class of binding sites, n = 0.060 mumol/mg with binding constant K = 2.34 x 10(4) M-1. The possibility of side effects caused by methotrexate-melanin interactions after treatment of neoplasms is discussed.

Model neuromelanins as antioxidative agents during lipid peroxidation.

The oxidative pathway of dopamine metabolism in the human brain leads to formation and accumulation of neuromelanin in the cytoplasm of most nigrostriatal dopaminergic neurons. The physiological significance of neuromelanin and its contribution to the neurodegenerative processes underlying Parkinson's disease are still controversial. The effect of model neuromelanins on Fe(II)/ascorbate-induced lipid peroxidation in micelles of linoleic acid and in lecithin liposomes was determined. Synthetic neuromelanins were obtained from dopamine (DA), 5-S-cysteinyldopamine (CysDA) or from equimolar mixture of these precursors. Thiobarbituric acid test and reverse-phase HPLC, used for measurements of primary and secondary oxidation products, showed that all melanins tested significantly suppressed peroxidation of both, linoleic acid and liposomal lecithin. The inhibitory effect of CysDA-melanin was lower than of DA/CysDA-melanin and DA-melanin. All the melanins were able to reduce linoleic acid hydroperoxides to their stable hydroxy derivatives. The results obtained suggest that neuromelanin can act as natural antioxidant. The fatty acid hydroperoxide-reducing ability demonstrated for the model neuromelanins appears to be involved in the mechanism of antioxidative activity of neuromelanin.

Free radical scavenging by ethanol extract of propolis.

The free radical scavenging ability of an ethanolic extract of propolis (EEP) was demonstrated by electron spin resonance spectroscopy, when 2,2-diphenyl-1-picrylhydrazyl (DPPH) was treated with increasing concentrations of EEP. It was shown that the DPPH signal intensity was inversely related to the EEP concentration and to the reaction time. It is assumed that the ability of components in EEP to donate a hydrogen atom is responsible for the lowering of the DPPH-EEP signal, and reflect the anti-oxidative nature of EEP.

Genetic disregulation of gene coding tumor necrosis factor alpha receptors (TNF alpha Rs) in follicular thyroid cancer--preliminary report.

UNLABELLED: TNF alpha receptors participate in programmed cell death. TNF R2 efficiently assists TNF R7 effects by ligand passing. Structure of TNF alpha receptors influences TNF activity in vivo and the structure of TNF R2 gene may suggest post-transcription modification based on alternative splicing. The aim of the study was to analyse the expression of gene coding TNF alpha receptors R2 and R7 by estimation of mRNA expression of differentiated thyroid carcinomas in comparison to surrounding tissue free from neoplastic infiltration and search for differently spliced TNF alpha R2/R7 isophorms. The study included seven patients with histopathologically confirmed follicular thyroid cancer. Tissue samples removed from tumor region were obtained from the follicular thyroid cancer patients undergoing surgical treatment. The samples were divided into two parts. The first one was routinely examined histopathologically, the second was used for RNA extraction and the number of TNF and its receptors mRNA copies were subsequently quantified. RESULTS: 1) The presence of TNF alpha expression was observed in all examined samples, in contrast to TNF R1 expression; 2) The high level of TNF alpha expression was noted both for typical and sought TNF R2/R7 isoforms and 3) A considerable number of samples displayed higher levels of TNF R2 isoforms than TNF R2/R7 mRNA expression. Genetic disregulation observed in neoplastic disease usually concerns dysfunction of cytokines receptor genes.

The inhibiting effect of catecholamine-melanins on UV-induced lecithin peroxidation.

It was found that the yield of thiobarbituric acid reactive substances in UV-irradiated liposome membranes was significantly suppressed in the presence of catecholamine-melanins, indicating their ability to inhibit lipid peroxidation. The extent of inhibition depended on the type and concentration of melanin polymers. Melanin-copper complexes inhibited lecithin photooxidation less effectively than copper-free melanins derived from the same precursor. The antioxidant efficiency of melanins appears to be related to the levels of intrinsic and photo-induced free radical centers in the melanin polymer, as well as to accessibility of these centers for active species formed during irradiation of liposomes.

Efficiency of lipofection of adherent cells is limited by apoptosis.

Stability of gene expression and transfection efficiency plays the main role in the application of gene transfer method. In somatic cell gene delivery, expression of the gene product is limited by the function of the cell to which it is delivered. In the present study analyzing the lipofected adherent cells, we have shown that lower level of transgene: beta-galactosidase activity at later time period correlated with decrease in cell viability, which was shown to be due to apoptosis. Apoptosis following DNA uptake occurred only when DNA was present during lipofection.

Differentiation strategies of alternative splicing variants mRNA estrogen receptor-alpha.

From the clinical point of view recognizing the concentrations and type profile of isoforms could be of significant practical importance. The aim of the study is designed QRT-PCR reaction to assess profile of mRNA ER-alpha and their isoforms. Theoretical part of the study was made according to computer program and available Genbank database. To detect a isoform one of the primer was designed to hybridize within exon-border linked in alternative splicing. The study presents the differentiation strategies in isoforms coming about as the alternative splicing result. Designed oligonucleotide probes and primers allow to distinguish mRNA isoforms of ER-alpha and their quantification in assessed tissues.

Quantitative analysis of estrogen receptor mRNA in human endometrium throughout the menstrual cycle using a real-time reverse transcription-polymerase chain reaction assay.

We conducted a quantitative analysis of ERalpha and ERbeta mRNA expression in normal human endometrium throughout the menstrual cycle in regular menstruating premenopausal women, taking advantage of this real-time PCR assay. Endometrial dating was determined from the histology of the endometrium and classified into: proliferative endometrium and secretory endometrium. Both ERalpha and ERbeta mRNA expression were detected in all endometrial samples at both proliferative and secretion phase. However ERalpha mRNA expression level was higher than that of ERbeta specially during proliferative phase. These results suggest that estrogenic effects occur predominantly through ERalpha than ERbeta.