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Infrared (IR) spectroscopy, atomic force microscopy (AFM), and dielectric spectroscopy methods were employed to study structural and dynamic changes in the tannic acid (TA)-stabilized pericardium tissue. Chemically stabilized pericardium tissue is widely used in construction of the tissue derived bioprostheses. IR spectra recorded in the range 400-4000 cm-1 allowed us to recognize different types of TA-collagen interactions. Formation of hydrogen bonds between amine as well as amide NH groups from collagen and hydroxyl groups of TA was analyzed. The AFM imaging showed that the stabilization procedure with TA introduces considerable changes in both surface topography and thickness of collagen fibrils as well as in fibril arrangement on the tissue surface. It was found, that these structural changes have an impact on the dielectric behavior of the TA-stabilized tissue. The dielectric spectra for the native and TA-stabilized tissues were measured in the frequency and temperature ranges of 10(-1) -10(7) Hz and 120-270 K, respectively. The dielectric spectra revealed the relaxation process due to orientation of bound water supplemented by the fluctuation of collagen polar side groups. At the temperatures above approximately 210 K, the relaxation due to ion migration process was observed. It was found that both relaxation processes were influenced by the TA-collagen interaction.

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INTRODUCTION: Structural modification of proteins, mainly collagen in connective tissues, is important in the manufacture of tissue-derived biomaterials. Natural compounds like genipin or tannic acid (TA) have been proposed instead of glutaraldehyde which shows cytotoxic effects on the processed tissue. Furthermore, calcification of glutaraldehyde-treated tissue limits the functional lifetime of bioprostheses. TA is known to form numerous hydrogen bonds with proteins. The purpose of our study was to investigate structural changes in porcine pericardium upon chemical modification with tannic acid. METHODS: Porcine pericardium tissue (PP) was soaked in 2% TA for 4, 24 or 48 hours. Changes in tissue structure were studied using electrophoresis (SDS-PAGE) and histological examination. Structural stability of PP tissue was evaluated by SDS/NaCl extraction method and enzymatic digestion with pancreatin. RESULTS: TA-modification of PP caused a time-dependent decrease in the number of peptides extracted from tissue. Microscopic studies revealed no significant morphological differences between native and TA-modified tissues, except for the native pancreatin-digested tissue where lack of both cells and low molecular peptides was observed. CONCLUSION: Modification of PP with TA causes the structural changes leading to an increase in the tissue resistance to SDS/NaCl extraction and enzymatic digestion, providing experimental evidence for the higher structural stability of TA-treated tissue.

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Native and chemically stabilized porcine pericardium tissue was imaged by the contact mode atomic force microscopy (AFM), in air. Chemically stabilized pericardium is used as a tissue-derived biomaterial in various fields of the reconstructive and replacement surgery. Collagen type I is the main component of the fibrous layer of the pericardium tissue. In this study, the surface topography of collagen fibrils in their native state in tissue and after chemical stabilization with different cross-linking reagents: glutaraldehyde (GA), dimethyl suberimidate (DMS) and tannic acid (TA) was investigated. It has been found that chemical stabilization causes considerable changes in the surface topography of collagen fibrils as well as in the spatial organization of the fibrils within the tissue. The observed changes in the D-spacing pattern of the collagen fibril correspond to the formation of intrafibrilar cross-links, whereas formation of interfibrilar cross-links is mainly responsible for the observed tangled spatial arrangement of fibrils and crimp structure of the tissue surface. The crimp structure was distinctly seen for the GA cross-linked tissue. Surface heterogeneity of the cross-linking process was observed for the DMS-stabilized tissue. SDS-PAGE electrophoresis was performed in order to evaluate the stabilization effect of the tissues treated with the cross-linking reagents. It has been found that stabilization with DMS, GA or TA enhances significantly the tissue resistance to SDS/NaCl extraction. The relation between the tissue stability and changes in the topography of the tissue surface was interpreted in terms of different nature of cross-links formed by DMS, GA and TA with collagen.

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Susceptibility to several antibiotics and biochemical properties of intestinal and soil strains of Desulfovibrio desulfuricans bacteria were investigated using the tests: ATB ANA, Sceptor Anaerobic MIC/ID and API ZYM. It was demonstrated that the D. desulfuricans strains were resistant to penicillin, cefoxitin, clindamycin, metronidazole, erythromycin, rifampicin and teicoplanin. The strains initially susceptible to imipenem became resistant to this drug following 72 h incubation with it. Of 25 analyzed antibiotics there was none that after 72 h action on the bacteria was effective in relation to all of the investigated strains. The differences in susceptibility of D. desulfuricans strains to antibiotics were not associated with the strains' biochemical properties.

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In presented study, differences among six intestinal Desulfovibrio desulfuricans strains were investigated regarding their ability of sulphasalazine (SAS) biotransformation. Bacterial strains were incubated (24 or 48 h) in liquid medium supplemented with SAS (12.2-150 microM). It has been demonstrated that investigated D. desulfuricans strains converted SAS with various rates, depending on the strain used and on time of drug contact with bacterial cells. Significantly different (50% coefficient of variability) were the maximum rates of SAS biotransformation. It was demonstrated that these rates were strongly dependent on the drug concentration. Increasing SAS concentrations up to about 120 microM caused increase in the drug transformation rate. At higher SAS concentrations this rate remained at the same level or was inconsiderable lower. The strains exposed to SAS for 24 hs transformed this drug with the rate about twice as compared with that observed after 48 h exposition.

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A simple method of whole intestinal crypts isolation from rat's colonic tissue has been developed. Culture of viable epithelial cells (colonocytes) was obtained from intact crypts using method providing colonocytes for apoptosis. Satisfactory results have been obtained if the crypts were isolated using collagenase I. Under conditions applied, spontaneous release of the colonocytes took place. Liberated cells underwent almost immediate adhesion to microcarrier surface. The primary culture of normal colonocytes indicating metabolic activity for long time (> 10 days) has been obtained.

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Various genera of sulphate reducing bacteria (SRB) have been found in the human digestive tract. It is suggested that some of SRB species may be responsible for the development of the clinical symptoms of ulcerative colitis and other disease of large intestine. Sulphasalazine (salicyl-azo-sulphapyridine, SAS) is commonly used to treat patients with ulcerative colitis and Crohn disease. Above 30 samples of faeces or biopsy specimens from 25 patients (age 45 +/- 14 years; M/F, 13/12) suffering from gastrointestinal disorders were used for isolation of Desulfovibrio desulfuricans species. The morphological, physiological and biochemical characteristics of isolated strains and also their susceptibility to SAS was determined. D. desulfuricans isolates were obtained from 5 amongst all patients assayed. Some abnormal, cigar-shaped cells were detected as accompanying the cells represented by rods, curved rods and vibrios. After strains purification, two types of colonies were present on the solid Postgate's medium B (containing lactate as a carbon source and sulphate for energy conservation): the black colonies growing in bulk of agar medium and the transparent, surface-growing mucous colonies. These two types of D. desulfuricans colonies may be a result of different iron availability for bacterial cells. High metabolic activity of strain was not always accompanied by the presence of H2S gas lock in the test tube, although the H2S odor was perceptible. All tested strains multiplied inconsiderably slowly in the presence of SAS at concentrations 10, 20, 40 and 60 mg/cm3. The growing concentrations of SAS did not cause a proportional decrease of the bacterial cells number. Taking into account the positive results of using SAS to treat patients with some colonic diseases and the indicated resistance to SAS of intestinal D. desulfuricans strains, it appears probable, that this SRB species isn't responsible for the development of mentioned diseases.

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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).

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A comparison of cellular fatty acid profiles of Desulfovibrio desulfuricans DSM 642 and 14 wild strains of this species, isolated from two completely different environments, soil and the human intestine, was carried out. All the D. desulfuricans strains grown on lactate and sulfate indicated the presence of considerable amounts of i-C15:0, i-C17:1 and C16:0. Although differences in the quantities of individual fatty acids present in each strain were clear in the group of soil strains (similarity, 67.6%), in contrast to almost identical fatty acid patterns (similarity, near 100%) in the intestinal strains, the results were variable within the limits acceptable for species demonstration. The higher similarity of the fatty acid profiles of intestinal strains may be a result of the similarity of biocenoses in the human digestive tract. The coefficients of variability of i-C17:1 and i-C15:0 (the major branched-chain fatty acids), as well as clustering of the investigated strains compared with strains described in the literature after plotting percentages of i-C17:1 fatty acid against i-C15:0 fatty acid, confirmed a certain heterogeneity of cellular fatty acid profiles within the group of soil strains, in contrast to almost ideal homogeneity within the group of intestinal isolates. Intestinal strains contained a higher ratio of saturated to unsaturated fatty acids (2.2 +/- 0.14) than did soil strains (1.6 +/- 0.2; in one case, 2.7). We propose that intestinal D. desulfovibrio bacteria should be assumed to be a highly homogeneous group and should be represented by the strain D. desulfuricans subsp. intestinus in collections of microbial cultures.

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