Effects of dietary flaxseed on vascular contractile function and atherosclerosis during prolonged hypercholesterolemia in rabbits.

Dietary flaxseed has significant anti-atherogenic effects. However, the limits of this action and its effects on vascular contractile function are not known. We evaluated the effects of flaxseed supplementation on atherosclerosis and vascular function under prolonged hypercholesterolemic conditions in New Zealand White rabbits assigned to one of four groups for 6, 8, or 16 wk of feeding: regular diet (RG), 10% flaxseed-supplemented diet (FX), 0.5% cholesterol-supplemented diet (CH), and 0.5% cholesterol- and 10% flaxseed-supplemented diet (CF). Cholesterol feeding resulted in elevated plasma cholesterol levels and the development of atherosclerosis. The CF group had significantly less atherosclerotic lesions in the aorta and carotid arteries after 6 and 8 wk than the CH animals. However, the anti-atherogenic effect of flaxseed supplementation was completely attenuated by 16 wk. Maximal tension induced in aortic rings either by KCl or norepinephrine was not impaired by dietary cholesterol until 16 wk. This functional impairment was not prevented by including flaxseed in the high-cholesterol diet. Aortic rings from the cholesterol-fed rabbits exhibited an impaired relaxation response to acetylcholine at all time points examined. Including flaxseed in the high-cholesterol diet completely normalized the relaxation response at 6 and 8 wk and partially restored it at 16 wk. No significant changes in the relaxation response induced by sodium nitroprusside were observed in any of the groups. In summary, dietary flaxseed is a valuable strategy to limit cholesterol-induced atherogenesis as well as abnormalities in endothelial-dependent vasorelaxation. However, these beneficial effects were attenuated during prolonged hypercholesterolemic conditions.

Role of intraoperative cholangiography during endoscopic cholecystectomy.

Six hundred sixty-nine German hospitals participated in a survey on the use of intraoperative cholangiography (IOC) in its relationship to common bile duct injuries. Of these, 174 surgical departments are presently practicing IOC, 30 routinely. Only 16 injuries to the bile duct occurred in the 6,328 patients who underwent surgery in those 30 units. Approximately 80% of these injuries were discovered during surgery because of IOC and were treated immediately. A significant difference was found between these rates and those reported by units that performed IOC selectively. Only 58% of bile duct injuries were discovered at the time of surgery in units performing selective cholangiography. In addition, it appears that IOC helps to avoid bile duct injuries; the 30 units that performed routine IOC were defined as "learners," as they had performed fewer LCs, and yet they had a significant lower bile duct injury rate than "experienced" centers that did not perform IOC routinely.

[Bile composition and antibiotic excretion. Observations with T-drainage]. / Gallenzusammensetzung und Antibiotikaausscheidung. Beobachtungen an der T-Drainage.

Patients comparable in disease, therapy and serum bilirubin concentration were either treated with mezlocillin intravenously or not at all. The bile of each patient was collected either from a T-drainage or from a percutaneously placed drainage into the bile ducts. The concentrations of GGT and AP, which were liberated by destroyed liver cells, and of bilirubin and mezlocillin, which were secreted actively, were analysed. Those patients who had normal serum bilirubin concentrations had a significantly higher biliary bilirubin excretion than those with high serum bilirubin level. The maximum excretion was after 4 hours. While the biliary concentration of bilirubin decreased, the concentration of secreted mezlocillin increased. Due to destroyed liver cells those patients with pathologically elevated blood bilirubin levels had a 50-fold lower mezlocillin excretion than those with normal blood values.

Polymerase chain reaction-aided genomic sequencing of an X chromosome-linked CpG island: methylation patterns suggest clonal inheritance, CpG site autonomy, and an explanation of activity state stability.

The 5' region of the gene encoding human X chromosome-linked phosphoglycerate kinase 1 (PGK1) is a promoter-containing CpG island known to be methylated at 119 of 121 CpG dinucleotides in a 450-base-pair region on the inactive human X chromosome in the hamster-human cell line X8-6T2. Here we report the use of polymerase chain reaction-aided genomic sequencing to determine the complete methylation pattern of this region in clones derived from X8-6T2 cells after treatment with the methylation inhibitor 5-azacytidine. We find (i) a clone showing full expression of human phosphoglycerate kinase is fully unmethylated in this region; (ii) clones not expressing human phosphoglycerate kinase remain methylated at approximately 50% of CpG sites, with a pattern of interspersed methylated (M) and unmethylated (U) sites different for each clone; (iii) singles, defined as M-U-M or U-M-U, are common; and (iv) a few CpG sites are partially methylated. The data are interpreted according to a model of multiple, autonomous CpG sites, and estimates are made for two key parameters, maintenance efficiency (Em approximately 99.9% per site per generation) and de novo methylation efficiency (Ed approximately 5%). These parameter values and the hypothesis that several independent sites must be unmethylated for transcription can explain the stable maintenance of X chromosome inactivation. We also consider how the active region is kept free of methylation and suggest that transcription inhibits methylation by decreasing Em so that methylation cannot be maintained. Thus, multiple CpG sites, independent with respect to a dynamic methylation system, can stabilize two alternative states of methylation and transcription.

In vivo footprint and methylation analysis by PCR-aided genomic sequencing: comparison of active and inactive X chromosomal DNA at the CpG island and promoter of human PGK-1.

The promoter region of the X-linked human phosphoglycerate kinase-1 (PGK-1) gene is a CpG island, similar to those often found near autosomal genes. We used ligation-mediated polymerase chain reaction (PCR) for a genomic sequencing study in which 450 bp of the human PGK-1 promoter region was analyzed for the presence of in vivo protein footprints and cytosine methylation at all CpG sites. A technique was devised to selectively visualize the DNA of the inactive X chromosome (Xi), even in the presence of the active X chromosome (Xa). We found that the human Xa in both normal male lymphocytes and hamster-human hybrids is completely unmethylated at all 120 CpG sites. In contrast, 118 of the CpG sites are methylated on the human Xi in hamster-human hybrids. The Xi in normal female lymphocytes is also highly methylated, but some GCG or CGC trinucleotides partially escape methylation; all other CpGs are fully methylated. In vivo footprinting studies with dimethylsulfate (DMS) revealed eight regions of apparent protein-DNA contacts on the Xa. Four of the footprints contained the consensus sequence of the binding site for transcription factor Sp1. The other regions include potential binding sites for transcription factors ATF, NF1, and a CCAAT-binding protein. The Xi did not show any specifically protected sequences, and with the exception of four hyperreactive sites, the in vivo DMS reactivity profile of Xi DNA was very similar to that of purified, linear Xi DNA. The implications of these findings with regard to the maintenance of methylation-free islands, X chromosome inactivation, and the chromatin structure of facultative heterochromatin are discussed.

Ligation-mediated PCR improves the sensitivity of methylation analysis by restriction enzymes and detection of specific DNA strand breaks.

DNA methylation at specific sites is most frequently studied by use of methylation-sensitive restriction endonucleases and Southern blotting. We report here that the sensitivity of this method can be increased several-hundred-fold by applying a ligation-mediated polymerase chain reaction (LM-PCR) procedure following enzyme treatment. DNA is cleaved simultaneously with two restriction enzymes, one sensitive and one insensitive to methylation. After cleavage, a gene-specific oligonucleotide primer is used for primer extension, followed by linker ligation and then conventional PCR. Using this technique, we demonstrate that DNA from 100 cells (about 0.6 ng) can be prepared and qualitatively analyzed for methylation at sites in an X-linked CpG island, and 50 ng of DNA can be analyzed quantitatively. A site 23 bp downstream of the major transcription start site of human phosphoglycerate kinase-1 (PGK-1) is 52 +/- 7 percent methylated in DNA from female blood and greater than 98 percent unmethylated in DNA from male blood or HeLa cells. This method detects quantitatively specific breaks in either double stranded or single stranded DNA. Thus new assays for enzymes and DNA structure can be devised.

Genomic sequencing and methylation analysis by ligation mediated PCR.

Genomic sequencing permits studies of in vivo DNA methylation and protein-DNA interactions, but its use has been limited because of the complexity of the mammalian genome. A newly developed genomic sequencing procedure in which a ligation mediated polymerase chain reaction (PCR) is used generates high quality, reproducible sequence ladders starting with only 1 microgram of uncloned mammalian DNA per reaction. Different sequence ladders can be created simultaneously by inclusion of multiple primers and visualized separately by rehybridization. Relatively little radioactivity is needed for hybridization and exposure times are short. Methylation patterns in genomic DNA are readily detectable; for example, 17 CpG dinucleotides in the 5' region of human X-linked PGK-1 (phosphoglycerate kinase 1) were found to be methylated on an inactive human X chromosome, but unmethylated on an active X chromosome.

The DNA methylation system in proliferating and differentiated cells.

The human melanoma cell line M21 can be induced to differentiate into oligodendrocyte-like cells with concommitant cessation of cell division. Cytosine-arabinoside, 5-aza-2'-deoxycytidine, hydroxyurea, aphidicolin, and phorbol-12-myristate-13-acetate were found to be potent differentiation inducers. We have analyzed the changes of methylation of DNA cytosines that occur after treatment of M21 cells with these compounds. Although DNA methylation levels remain unchanged in the presence of aphidicolin and phorbol ester, 5-aza-2'-deoxycytidine-induced differentiation of these cells results in a 40% DNA demethylation. On the other hand, hydroxyurea and cytosine-arabinoside treatment causes DNA hypermethylation, which, in the case of the cytidine analogue is of only transient nature. These results show that the differentiation of human melanoma cells can be accompanied by variable changes of DNA methylation levels. In another set of experiments, the DNA methylation levels have been analyzed during cytosine-arabinoside-induced differentiation of human K562 erythroleukemia cells. In this system, a transient DNA demethylation precedes the establishment of the differentiated phenotype. Since DNA replication is inhibited, this demethylation cannot be explained by inhibition of the maintenance activity of DNA methyltransferase, but is more likely caused by an active excision of 5-methylcytosine from DNA.

Variable DNA methylation changes during differentiation of human melanoma cells.

The DNA 5-methylcytosine content has been analyzed in the human melanoma cell line M21 at several time points after induction of differentiation by a variety of inducers. 5-Aza-2'-deoxycytidine reduces DNA methylation to about 50% of the control level and this demethylation occurs prior to the establishment of the differentiated phenotype. The DNA synthesis inhibitors cytosine arabinoside, aphidicolin, and hydroxyurea exert different effects on DNA methylation in these cells. Cytosine arabinoside induces an early DNA hypermethylation, which is however reversible and drops to the original level after 24 h. Hydroxyurea induces DNA hypermethylation after a lag period of more than 48 h and the DNA polymerase alpha inhibitor aphidicolin has no effect on the DNA methylation level. Treatment of cells with phorbol 12-myristate 13-acetate, another potent inducer of melanoma cell differentiation, does not result in a change of total DNA methylation over a period of 96 h. These results indicate that differentiation of human melanoma cells can be accompanied by variable changes of the DNA methylation pattern. These changes can be neither generally related to the differentiation process itself nor related to the effects of DNA synthesis inhibition on DNA methylation, but may more likely reflect a direct or indirect particular effect of the inducer on the DNA methylation process.

DNA methylation levels in acute human leukemia.

We have studied the overall 5-methylcytosine content and the percentage of methylated CpG-dinucleotides in 25 cases of untreated human acute leukemias. For comparison, normal leucocyte subpopulations were similarly analyzed. The methylation levels in normal white blood cell DNA varied in the same range as those in leukemia cells with no apparent hypomethylation in tumor cell DNA. Such hypomethylation, however, was found in a patient studied in first and second relapses of the disease. These data suggest that genome-wide demethylation, a characteristic of other tumors, does not accompany leukemic transformation.