Global Demethylation of Rat Chondrosarcoma Cells after Treatment with 5-Aza-2′-Deoxycytidine Results in Increased Tumorigenicity

Abnormal patterns of DNA methylation are observed in several types of human cancer. While localized DNA methylation of CpG islands has been associated with gene silencing, the effect that genome-wide loss of methylation has on tumorigenesis is not completely known. To examine its effect on tumorigenesis, we induced DNA demethylation in a rat model of human chondrosarcoma using 5-aza-2-deoxycytidine. Rat specific pyrosequencing assays were utilized to assess the methylation levels in both LINEs and satellite DNA sequences following 5-aza-2-deoxycytidine treatment. Loss of DNA methylation was accompanied by an increase in invasiveness of the rat chondrosarcoma cells, in vitro, as well as by an increase in tumor growth in vivo. Subsequent microarray analysis provided insight into the gene expression changes that result from 5-aza-2-deoxycytidine induced DNA demethylation. In particular, two genes that may function in tumorigenesis, sox-2 and midkine, were expressed at low levels in control cells but upon 5-aza-2-deoxycytidine treatment these genes became overexpressed. Promoter region DNA analysis revealed that these genes were methylated in control cells but became demethylated following 5-aza-2-deoxycytidine treatment. Following withdrawal of 5-aza-2-deoxycytidine, the rat chondrosarcoma cells reestablished global DNA methylation levels that were comparable to that of control cells. Concurrently, invasiveness of the rat chondrosarcoma cells, in vitro, decreased to a level indistinguishable to that of control cells. Taken together these experiments demonstrate that global DNA hypomethylation induced by 5-aza-2-deoxycytidine may promote specific aspects of tumorigenesis in rat chondrosarcoma cells.     

Perceived wellbeing of patients one year post stroke in general practice - <Emphasis Type="Italic">recommendations for quality aftercare</Emphasis>

Background  

Annually, 41,000 people in the Netherlands have strokes. This has multiple physical and psychosocial consequences. Most patients return home after discharge from hospital. Quality aftercare by general practitioners is important to support patients at home. The purpose of this study is to examine the wellbeing of patients who returned home immediately after discharge from hospital, one year post stroke, in comparison with the general Dutch population of the same age and to determine factors that could influence wellbeing.     

The Free-Radical Nature of Nitric Oxide Molecules as a Determinant of their Conversion to Nitrosonium Cations in Living Systems

Biophysics - This paper presents new results that confirm our previous inference that the binuclear form of biologically active dinitrosyl iron complexes (B-DNICs) with thiol-containing ligands...     

Dinitrosyl Iron Complexes with Thiol-Containing Ligands Can Suppress Viral Infections as Donors of the Nitrosonium Cation (Hypothesis)

Biophysics - The appropriateness of verification of the possible antiviral effect of dinitrosyl iron complexes with thiol-containing ligands as donors of nitrosonium cations (NO+) is argued. There...     

Mechanism of inhibition of catalase by nitro and nitroso compounds

Dinitrosyl iron complexes (DNIC) with thiolate ligands and S-nitrosothiols, which are NO and NO+ donors, share the earlier demonstrated ability of nitrite for inhibition of catalase. The efficiency of inhibition sharply (by several orders in concentration of these agents) increases in the presence of chloride, bromide, and thiocyanate. The nitro compounds tested--nitroarginine, nitroglycerol, nitrophenol, and furazolidone--gained the same inhibition ability after incubation with ferrous ions and thiols. This is probably the result of their transformation into DNIC. None of these substances lost the inhibitory effect in the presence of the well known NO scavenger oxyhemoglobin. This fact suggests that NO+ ions rather than neutral NO molecules are responsible for the enzyme inactivation due to nitrosation of its structures. The enhancement of catalase inhibition in the presence of halide ions and thiocyanate might be caused by nitrosyl halide formation. The latter protected nitrosonium ions against hydrolysis, thereby ensuring their transfer to the targets in enzyme molecules. The addition of oxyhemoglobin plus iron chelator o-phenanthroline destroying DNIC sharply attenuated the inhibitory effect of DNIC on catalase. o-Phenanthroline added alone did not influence this effect. Oxyhemoglobin is suggested to scavenge nitrosonium ions released from decomposing DNIC, thereby preventing catalase nitrosation. The mixture of oxyhemoglobin and o-phenanthroline did not affect the inhibitory action of nitrite or S-nitrosothiols on catalase.     

Effect of dinitrosyl iron complexes with thiol-containing ligands on the penile cavernosum bodies in rats

A beneficial effect of dinitrosyl iron complexes (DNIC) with thiol-containing ligands on penile cavernus tissue was shown in rats subjected to penile denervation. Histological and histochemical investigations demonstrated that intracavernous injections of dinitrosyl iron complexes (2 times per one week during 6 months) blocked the reinforcement of endothelial cell proliferation in the tissue characteristic of the cavernous tissue when the penile nerve was removed. On the other hand, treatment with dinitrosyl iron complexes led to the preservation of mitotic activity of smooth myocytes and protected against the appearance in these cells of collagenase, an indicator of muscle transformation into fibrous tissue. It was shown that the process of fibrous transformation of myocytes correlates with a decrease in the mitotic activity of fibroblasts in the adventive part of cavernosa. The mitotic activity increased in cavernous tissue in the absence of dinitrosyl iron complexes. The efficiency of long-term action of dinitrosyl iron complexes on the erection in both intact animals and animals subjected to neuroectomy of cavernous tissue nerve was shown. The injection of low-molecular dinitrosyl iron complexes to the cavernous tissue resulted in the formation of protein-bound dinitrosyl iron complexes in the tissue, which were detected by the EPR technique. It is assumed that these dinitrosyl iron complexes function as a depot of nitric oxide, providing long-lasting penis erection.     

A microsatellite linkage map of Drosophila mojavensis

Background

Drosophila mojavensishas been a model system for genetic studies of ecological adaptation and speciation. However, despite its use for over half a century, no linkage map has been produced for this species or its close relatives.

Results

We have developed and mapped 90 microsatellites in D. mojavensis, and we present a detailed recombinational linkage map of 34 of these microsatellites. A slight excess of repetitive sequence was observed on the X-chromosome relative to the autosomes, and the linkage groups have a greater recombinational length than the homologous D. melanogaster chromosome arms. We also confirmed the conservation of Muller's elements in 23 sequences between D. melanogaster and D. mojavensis.

Conclusions

The microsatellite primer sequences and localizations are presented here and made available to the public. This map will facilitate future quantitative trait locus mapping studies of phenotypes involved in adaptation or reproductive isolation using this species.     

Evolution of the sulfide-binding function within the globin multigenic family of the deep-sea hydrothermal vent tubeworm Riftia pachyptila

The giant extracellular hexagonal bilayer hemoglobin (HBL-Hb) of the deep-sea hydrothermal vent tube worm Riftia pachyptila is able to transport simultaneously O(2) and H(2)S in the blood from the gills to a specific organ: the trophosome that harbors sulfide-oxidizing endosymbionts. This vascular HBL-Hb is made of 144 globins from which four globin types (A1, A2, B1, and B2) coevolve. The H(2)S is bound at a specific location (not on the heme site) onto two of these globin types. In order to understand how such a function emerged and evolved in vestimentiferans and other related annelids, six partial cDNAs corresponding to the six globins known to compose the multigenic family of R. pachyptila have been identified and sequenced. These partial sequences (ca. 120 amino acids, i.e., 80% of the entire protein) were used to reconstruct molecular phylogenies in order to trace duplication events that have led to the family organization of these globins and to locate the position of the free cysteine residues known to bind H(2)S. From these sequences, only two free cysteine residues have been found to occur, at positions Cys + 1 (i.e., 1 a.a. from the well-conserved distal histidine) and Cys + 11 (i.e., 11 a.a. from the same histidine) in globins B2 and A2, respectively. These two positions are well conserved in annelids, vestimentiferans, and pogonophorans, which live in sulfidic environments. The structural comparison of the hydrophobic environment that surrounds these cysteine residues (the sulfide-binding domain) using hydrophobic cluster analysis plots, together with the cysteine positions in paralogous strains, suggests that the sulfide-binding function might have emerged before the annelid radiation in order to detoxify this toxic compound. Moreover, globin evolutionary rates are highly different between paralogous strains. This suggests that either the two globin subfamilies involved in the sulfide-binding function (A2 and B2) have evolved under strong directional selective constraints (negative selection) and that the two other globins (A1 and B1) have accumulated more substitutions through positive selection or have evolved neutrally after a relaxation of selection pressures. A likely scenario on the evolution of this multigenic family is proposed and discussed from this data set.