The prevalence of Chlamydia pneumoniae in the aortic wall and in peripheral blood of patients scheduled for coronary artery bypass grafting.

Some reports confirm a potential role of Chlamydia pneumoniae (ChP) in atherogenesis. In order to explore possible association between ChP and atherosclerosis, investigations were carried out in which the frequency of ChP in the arterial wall and peripheral blood was assessed in a group of patients with chronic coronary artery disease (CAD). Fifty-seven patients were enrolled in the study, 13 women and 44 men aged 61.8±6.5 (47-74), with previously diagnosed CAD, scheduled for planned coronary artery bypass grafting due to clinical indications. Vessel specimens retrieved from the ascending aorta (as a part of routine proximal venous graft development procedure) and peripheral blood mononuclear cells (PBMCs) from venous blood were evaluated for the presence of ChP DNA. Genomic DNA was extracted from PBMCs and vessel specimens. Quantitative real-time polymerase chain reaction (qPCR) was performed to detect ChP DNA. A statistically more frequent occurrence of ChP was observed in aortic tissues compared to blood samples (70.2% vs 56.1%, respectively). Similarly, the number of ChP DNA genomic copies [n/1µg genomic DNA] was significantly higher in tissue specimens compared to blood samples (89±91 vs 41±77, respectively; p=0.0046). In patients without ChP in blood specimens, we observed significantly higher amounts of ChP in tissue specimens compared to patients with ChP in blood specimens (156±71 vs 107±88, respectively; p=0.0453). No correlation was found between the number of ChP DNA copies [n/1µg genomic DNA] in blood and in aortic specimens. The infection of ChP in the aortic wall was connected with hypercholesterolemia (p=0.029) and diabetes (p=0.03). We conclude that Chlamydia pneumoniae is a pathogen frequently occurring in the aortic wall of patients with CAD. The occurrence of ChP DNA in the aortic tissue is related to classic CAD risk factors such as diabetes and dyslipidemia.

Hydralazine stimulates production of oxygen free radicals in Eagle's medium and cultured fibroblasts.

The effect of hydralazine on the oxygen free radical production was studied in whole cultured murine liver fibroblasts and mitochondrial and microsomal fractions of the cells by ESR spin trapping with DMPO and measurement of Tiron semiquinone formation. Hydralazine itself was found to generate free radicals in phosphate buffer and especially in Eagle's Minimal Essential Medium. Most of the adduct of the spin trap DMPO was due to its reaction with hydralazine-induced hydroxyl radical. Moreover, this compound stimulated free radical formation in fibroblasts. These data suggest that hydralazine alters the cellular free radical metabolism which may have implications for the biological activity of this drug.

Changes in DNA supercoiling in fibroblasts cultured in the presence of hydralazine.

We have analyzed changes in the supercoiling of nucleoid DNA of murine fibroblasts cultured in the presence of hydralazine. The entire DNA attached to the nuclear matrix was extracted from the cells and sedimented in neutral sucrose density gradients containing ethidium bromide. Nucleoids from cells treated with hydralazine responded to increasing ethidium bromide concentrations in a different way than those from control cultures. That is, supercoiled loops of DNA unwound with lower concentrations of ethidium bromide sedimented less rapidly than those of control cells, indicating that hydralazine reduced the degree of DNA supercoiling. Also, nucleoids from the drug-treated cells resisted the transition from relaxed to positive supercoiling at higher concentrations of ethidium bromide. Changes in nucleoid DNA supercoiling correlated directly with the dose of hydralazine in the fibroblast culture.

The delay of anoikis due to the inhibition of protein tyrosine dephosphorylation enables the maintenance of normal rat colonocyte primary culture.

Apoptosis induced by detachment of cells from the extracellular matrix (anoikis) appears to be one of the main obstacles in attempts to establish long-term primary culture of normal colonocytes. In the present study, the dynamics of molecular events related to apoptosis of isolated normal rat colonocytes was investigated. The whole colonic crypts were isolated using collagenase/dispase digestion technique. DNA fragmentation typical for the apoptosis and the apoptotic morphology of cells were observed already at the end of their isolation. Considerable increase in caspase-3 activity was noted during the first two hours of cell cultivation. Delaying of apoptosis by treatment of cells with sodium orthovanadate, the specific protein tyrosine phosphatase inhibitor, was found to be possible. It may facilitate long-term culture of intestinal epithelial cells.

The antioxidant enzymes activity in the conditions of systemic hypersilicemia.

The effect of an excessive inorganic silicon oral intake on the activity of basic antioxidant enzymes was studied in rats. Activities of superoxide dismutase, catalase, and glutathione peroxidase were measured in liver and kidney tissues of animals receiving per os sodium metasilicate nonahydrate (Na2SiO3.9H2O) (Sigma, [St. Louis, MO]) dissolved in their drinking water. A decrease of the activity of all the studied enzymes was found in the samples derived from the experimental group. The results obtained indicate the free oxygen radicals participation in the potential pathologic events in the conditions of systemic hypersilicemia.

Susceptibility to antibiotics and biochemical properties of Desulfovibrio desulfuricans strains.

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.

Quantitative evaluation of transcriptional activation of NF-κB p65 and p50 subunits and IκBα encoding genes in colon cancer cells by Desulfovibrio desulfuricans endotoxin.

Quantification of p65, p50 and IκBα mRNAs was performed by real time QRT-PCR in Caco-2 cells treated with 10, 50, and 100 µg/mL of Desulfovibrio desulfuricans LPS for 1, 6, 12, and 24 h. A strong increase in expression of p65 and IλBα genes was induced by 10 and 100 µg/mL of LPS at 1 h; after 6 h higher transcript amounts of both genes were observed at 100 µg/mL LPS. The p65 expression level was significantly increased by 50 and 100 µg/mL at 12 h and lowered by all LPS doses at 24 h. No significant differences between IκBα mRNA quantity in cells exposed to LPS at 12 and 24 h were observed. No changes in expression of p50 mRNA were induced by LPS. The expression of p65 gene positively correlated with IκBα gene expression. D. desulfuricans LPS is capable of modulating transcriptional activity of p65 and IκBα genes in intestinal epithelial cells.

[In vitro studies of the effect of growth factors on the connective tissue]. / Wplyw czynników wzrostowych na tkanke laczna w badaniach in vitro.

The paper presents recent data concerning the effect of growth factors on the metabolism of collagen, collagenases and glycosaminoglycans in vitro. The hypotheses of the mitogenic action of peptide factors and their role in the control of synthesis and breakdown of polyphosphoinositides are also reviewed.

Spin label detection and free radical nature of DNA damage by hydralazine.

1. Incubation with hydralazine was shown to induce degradative changes of calf thymus DNA spin-labeled with 3-(2-bromoacetamido)-2,2,5,5-tetramethyl-1-pyrrolidono-1-oxyl and 4-(2-bromoacetamido)-2,2,6,6-tetramethylpiperidino-1-oxyl detectable from electron spin resonance specta. 2. Hydralazine, especially in the presence of Fe2+ induced formation of thiobarbituric acid (TBA)-reactive DNA degradation products. 3. The formation of TBA-reactive products was prevented by catalase, EDTA and scavengers of .OH radicals and enhanced by superoxide dismutase which suggests that .OH radicals formed by the Fenton mechanism mediate the DNA damage by hydralazine-Fe2+.

Influence of hydrazinophthalazines on the activity of collagenase in murine fibroblast culture.

Murine fibroblasts cultured in vitro were treated with hydrazinophthalazine drugs inducing the lupus erythematosus-like syndrome. Collagenase enzyme activity in the growth medium was found to be increased 3-fold by hydralazine and 1.8-fold by binazine, as compared to the control cultures. The enhanced level of collagenolytic activity points to the metabolic changes in the connective tissue in response to hydrazinophtalazines.

Effect of anti-inflammatory drugs on the activity of antioxidant enzymes and in vivo peroxidation products in the liver and kidney of rat.

1. Four groups of rats were injected with four different anti-inflammatory drugs (indomethacin, phenylbutazone, acetylsalicylic acid and hydrocortisone) and the activities of superoxide dismutase, catalase and glutathione peroxidase were studied in the liver and kidney. 2. The drugs treatment resulted in decreased activity of the enzymes in both organs compared to the control animals. 3. In vivo tissue peroxidation was also effected by the drugs used. 4. Our results indicate that the changes of oxygen free-radical metabolism contribute to the action of these drugs in vivo.

Hyaluronic acid content and hyaluronidase activity in liver and spleen of rats with hydralazine-induced collagen-like syndrome.

A drug-induced collagen-like syndrome was produced in rats by hydralazine administration for 8, 9 and 10 months. The hyaluronic acid content and the hyaluronidase activity in liver and spleen were studied. A decrease in the hyaluronic acid amount was accompanied by an enhanced activity of hyaluronidase in both tissues. The results provide evidence for metabolic disturbances of glycosaminoglycans in the connective tissue in the collagen disease-like syndrome.

Usefulness of granulosa cells culture for testing of FSH activity.

The trials have been undertaken to elaborate the experimental conditions of granular cells cultivation for FSH activity testing.

Hyaluronic acid and other glycosaminoglycans of the human palatal tonsils.

The aim of this study was to determine whether the proportions of individual glycosaminoglycans (GAGs) change with bacterial infection of the human palatal tonsils and after recovery as compared with the physiological state of the palatal tonsils. In tonsillitis the greatest changes were related to hyaluronic acid (decrease) and heparin (increase). In the cured palatal tonsils the content of GAG components was found to return to that of the physiological state of the tonsils. The changes in GAG components in the cured palatal tonsils may possible contribute to the reversal of the human tonsillitis. The supposition is drawn that also physiological function of the cured tonsils is resumed.

Activities of antioxidant enzymes in fibroblasts cultured in vitro in the presence of hydralazine.

Variations in superoxide dismutase, catalase and glutathione peroxidase activity were studied in fibroblasts cultured in the presence of hydralazine, a drug known to be an inducer of the so-called collagen-like syndrome. The results demonstrated that both superoxide dismutase and catalase undergo a marked decrease in their activity, whereas glutathione peroxidase manifests a significant increase in its activity after treatment with hydralazine as compared to control cell cultures. Also the lipid peroxide concentration as expressed by the malondialdehyde amount was estimated in the above cultures. The altered antioxidant enzyme activity and the presence of byproducts of free radical damage support the possibility that the action of hydralazine leading to the pathogenesis of collagen disease-like syndrome involves an abnormal free-radical metabolism.

Polydispersity of glycosaminoglycan components in human palatal tonsils.

The aim of this study was to characterize glycosaminoglycan components in human palatal tonsils and to determine whether the proportions of individual glycosaminoglycans change with bacterial infection of the palatal tonsils and after recovery. The changes in glycosaminoglycans' content in the course of tonsillitis were mainly related to heparin and chondroitin-4-sulphate (increase) and hyaluronic acid (decrease). After recovery the polydispersity of glycosaminoglycans was found to be comparable to that in normal palatal tonsils.

Biochemical changes in cultured murine fibroblasts after treatment with hydrazinophthalazines.

Fibroblast cultures exposed to the drugs inducing a collagen-like syndrome (hydralazine and binazine) displayed growth inhibition and decrease in cellular protein content in a dose-dependent manner compared with control cultures. This was accompanied by the inhibitory effect of the drugs on DNA synthesis. The changes in the basic biochemical parameters of fibroblasts testify to the toxicity of hydrazinophthalazines in the connective tissue.

Single-strand breaks and repair in nuclear DNA in the presence of hydralazine assayed by the nucleoid technique.

The nucleoid sedimentation assay was used to study hydralazine-induced DNA structural changes and repair in the fibroblasts cultured in vitro. The drug induced a dose dependent loss in negative DNA supercoiling due to the physical breakage of the DNA. Relaxation of supercoiled DNA resulted in the nucleoids sedimenting with lower velocities than those of undamaged control cultures. Repair incubation of the cells did not cause the restoration of DNA supercoiling to control level. Unsuccessful repair of DNA damaged by hydralazine may result in maintaining the damaged DNA in the cell which could have immunologic consequences.

Effect of elastin peptides on the activities of antioxidant enzymes in fibroblasts.

The effect of elastin peptides (kappa-elastin) was investigated on murine fibroblasts. The data indicate that elastin peptides increase the activities of antioxidant enzymes detoxifying free radicals and increase the lipid peroxide concentration within the cell. These results suggest that the influence of elastin peptides on oxygen metabolism may be related to their activities in vivo following elastin degradation and can contribute to their role in the pathogenesis of atherosclerosis.

Properties of human red cell spectrin heterodimer (side-to-side) assembly and identification of an essential nucleation site.

The antiparallel side-to-side association of spectrin alpha and beta monomers is a two-step process which occurs in seconds even at 0 degrees C and at low concentrations. Assembly involves initial contact of complementary nucleation sites on each subunit, which are located near the actin binding end of the long, flexible heterodimer rod. The minimum nucleation sites are comprised of approximately four contiguous 106-residue homologous segments or repeats. Three repeats in the nucleation site contain an 8-residue insertion and have the highest homology to the four spectrin-like repeats in alpha-actinin. The adjacent actin binding domain on the beta subunit and the adjacent EF hand motifs on the alpha subunit are not required for heterodimer assembly. The nucleation sites probably have a specific lock and key structure which defines the unique side-to-side pairing of the many homologous segments in both subunits. Assembly of spectrin heterodimers is probably most analogous to a zipper. After initial nucleation site binding, the remainder of the subunits quickly associate along their full lengths to reconstitute a normal dimer by supercoiling around each other to form a rope-like, flexible rod. Assembly is terminated if either polypeptide is interrupted by a protease cleavage. Heterozygotic mutations involving either nucleation site are predicted to affect allele incorporation into the mature membrane skeleton.