The role of the DNA hypermethylating agent Budesonide in the decatenating activity of DNA topoisomerase II

Catenations between sister chromatids result from DNA replication and must be resolved to ensure proper chromatid segregation in mitosis. Functionally active Topoisomerase II (Topo II), through its mechanism of concerted breaking and rejoining of double stranded DNA, is required to carry out this fundamental process. In previous studies we have shown that modifications in DNA sequence by halogenated pyrimidines and by the demethylating agent 5-azacytidine leads to malfunction of Topo II that results in an increased yield of endorreduplicated cells as a result of segregation failure. In the present work we have evaluated the possible influence of the methylating agent Budesonide to modify the frequency of endoreduplicated cells in AA8 Chinese hamster cell population. Our results seem to indicate that when Budesonide was administered for two consecutive cell cycles did induce an increase in the yield of endoreduplicated cells, as previously observed for the hypomethylating agent 5-azaC. We have also examined the possible relationship between extensive hypermethylation induced by Budesonide in DNA and stabilization of cleavable complexes by m-AMSA. Taken as a whole, our results show that the degree of methylation in DNA correlates with the effectiveness of m-AMSA to stabilize the Topo II-DNA complexes and to induce DNA cleavage. These findings evidence for the first time the functional importance of DNA hyper- and hypomethylation changes as epigenetic factors able to modulate Topo II activity for proper chromosome segregation.     

Changes in respiratory mitochondrial machinery and cytochrome and alternative pathway activities in response to energy demand underlie the acclimation of respiration to elevated CO2 in the invasive Opuntia ficus-indica

Studies on long-term effects of plants grown at elevated CO(2) are scarce and mechanisms of such responses are largely unknown. To gain mechanistic understanding on respiratory acclimation to elevated CO(2), the Crassulacean acid metabolism Mediterranean invasive Opuntia ficus-indica Miller was grown at various CO(2) concentrations. Respiration rates, maximum activity of cytochrome c oxidase, and active mitochondrial number consistently decreased in plants grown at elevated CO(2) during the 9 months of the study when compared to ambient plants. Plant growth at elevated CO(2) also reduced cytochrome pathway activity, but increased the activity of the alternative pathway. Despite all these effects seen in plants grown at high CO(2), the specific oxygen uptake rate per unit of active mitochondria was the same for plants grown at ambient and elevated CO(2). Although decreases in photorespiration activity have been pointed out as a factor contributing to the long-term acclimation of plant respiration to growth at elevated CO(2), the homeostatic maintenance of specific respiratory rate per unit of mitochondria in response to high CO(2) suggests that photorespiratory activity may play a small role on the long-term acclimation of respiration to elevated CO(2). However, despite growth enhancement and as a result of the inhibition in cytochrome pathway activity by elevated CO(2), total mitochondrial ATP production was decreased by plant growth at elevated CO(2) when compared to ambient-grown plants. Because plant growth at elevated CO(2) increased biomass but reduced respiratory machinery, activity, and ATP yields while maintaining O(2) consumption rates per unit of mitochondria, we suggest that acclimation to elevated CO(2) results from physiological adjustment of respiration to tissue ATP demand, which may not be entirely driven by nitrogen metabolism as previously suggested.     

Cutaneous metastasis of occult hepatocellular carcinoma: a case report

BACKGROUND: Skin metastases from hepatocellular carcinoma (HCC) represented only 0.8% of all known cutaneous metastases in a recent large series. The most frequent site appears to be the head, but this fact has received little attention. An accurate cytologic diagnosis is extremely difficult in patients with unknown liver dysfunction. We report the cytologic features of a face metastasis from occult HCC. CASE: A 65-year-old woman presented with a mass in the right preauricular region of 2 months' duration. Her past medical history was noncontributory. Fine needle aspiration cytology was performed. Following the cytologic diagnosis, computed tomography revealed a 6-cm mass in the right lobe of the liver, portal vein thrombosis and involvement of the superior vena cava. The smears were very cellular. The most frequent pattern was trabecular with transmural endothelization. The cells had an epithelial appearance and polyhedral shape, exhibiting distinct borders. The nuclei were centrally placed, with a prominent nucleolus. The cytoplasm was granular. There were numerous atypical bare nuclei. Subsequent staining with antihepatocyte showed positivity in most tumor cells. The final diagnosis was metastatic HCC. CONCLUSION: HCCs should be considered in the differential diagnosis of carcinomas metastatic to the face, even in the absence of liver symptoms.     

Differential expression of the three multicopper oxidases from Myxococcus xanthus

Myxococcus xanthus is a soil bacterium that undergoes a unique life cycle among the prokaryotes upon starvation, which includes the formation of macroscopic structures, the fruiting bodies, and the differentiation of vegetative rods into coccoid myxospores. This peculiarity offers the opportunity to study the copper response in this bacterium in two different stages. In fact, M. xanthus vegetative rods exhibit 15-fold-greater resistance against copper than developing cells. However, cells pre-adapted to this metal reach the same levels of resistance during both stages. Analysis of the M. xanthus genome reveals that many of the genes involved in copper resistance are redundant, three of which encode proteins of the multicopper oxidase family (MCO). Each MCO gene exhibits a different expression profile in response to external copper addition. Promoters of cuoA and cuoB respond to Cu(II) ions during growth and development; however, they show a 10-fold-increased copper sensitivity during development. The promoter of cuoC shows copper-independent induction upon starvation, but it is copper up-regulated during growth. Phenotypic analyses of deletion mutants reveal that CuoB is involved in the primary copper-adaptive response; CuoA and CuoC are necessary for the maintenance of copper tolerance; and CuoC is required for normal development. These roles seem to be carried out through cuprous oxidase activity.     

Syntaxin and VAMP association with lipid rafts depends on cholesterol depletion in capacitating sperm cells

Sperm cells represent a special exocytotic system since mature sperm cells contain only one large secretory vesicle, the acrosome, which fuses with the overlying plasma membrane during the fertilization process. Acrosomal exocytosis is believed to be regulated by activation of SNARE proteins. In this paper, we identified specific members of the SNARE protein family, i.e., the t-SNAREs syntaxin1 and 2, and the v-SNARE VAMP, present in boar sperm cells. Both syntaxins were predominantly found in the plasma membrane whereas v-SNAREs are mainly located in the outer acrosomal membrane of these cells. Under non-capacitating conditions both syntaxins and VAMP are scattered in well-defined punctate structures over the entire sperm head. Bicarbonate-induced in vitro activation in the presence of BSA causes a relocalization of these SNAREs to a more homogeneous distribution restricted to the apical ridge area of the sperm head, exactly matching the site of sperm zona binding and subsequent induced acrosomal exocytosis. This redistribution of syntaxin and VAMP depends on cholesterol depletion and closely resembles the previously reported redistribution of lipid raft marker proteins. Detergent-resistant membrane isolation and subsequent analysis shows that a significant proportion of syntaxin emerges in the detergent-resistant membrane (raft) fraction under such conditions, which is not the case under those conditions where cholesterol depletion is blocked. The v-SNARE VAMP displays a similar cholesterol depletion-dependent lateral and raft redistribution. Taken together, our results indicate that redistribution of syntaxin and VAMP during capacitation depends on association of these SNAREs with lipid rafts and that such a SNARE-raft association may be essential for spatial control of exocytosis and/or regulation of SNARE functioning.     

Novel series of substituted biphenylmethyl urea derivatives as MCH-R1 antagonists for the treatment of obesity

We have designed and synthesized two novel series of MCH-R1 antagonists based on a substituted biphenylmethyl urea core. SAR was explored, suggesting that optimal binding with the receptor was achieved when the biphenylmethyl group and the linker were substituted on the same nitrogen of the urea moiety. Compound 1-(3'-cyano-4-biphenylmethyl)-3-(2-hydroxy-1,1-dimethylethyl)-1-{2-[1-(4-methylbenzyl)-4-piperidinyl]ethyl}urea 2t showed the best antagonist binding activity to the MCH-R1 with a 43 nM K(i).     

Staying out in the cold: glacial refugia and mitochondrial DNA phylogeography in ancient European brown bears

Models for the development of species distribution in Europe typically invoke restriction in three temperate Mediterranean refugia during glaciations, from where recolonization of central and northern Europe occurred. The brown bear, Ursus arctos, is one of the taxa from which this model is derived. Sequence data generated from brown bear fossils show a complex phylogeographical history for western European populations. Long-term isolation in separate refugia is not required to explain our data when considering the palaeontological distribution of brown bears. We propose continuous gene flow across southern Europe, from which brown bear populations expanded after the last glaciation.     

Eradication of Helicobacter pylori for the prevention of peptic ulcer rebleeding

AIM: To evaluate the effect of Helicobacter pylori eradication on ulcer bleeding recurrence in a prospective, long-term study including more than 400 patients. METHODS: Patients with peptic ulcer bleeding were prospectively included. H. pylori infection was confirmed by rapid urease test, histology or (13)C-urea breath test. Several eradication regimens were used. Ranitidine 150 mg was administered daily until eradication was confirmed by breath test 8 weeks after completing eradication therapy. Patients with therapy failure received a second or third course of therapy. Patients with eradication success did not receive maintenance anti-ulcer therapy, and were controlled yearly with a repeated breath test. RESULTS: Four hundred and twenty-two patients were followed up for at least 12 months, with a total of 906 patient-years of follow up. Mean age was 59 years, and 35% were previous nonsteroidal anti-inflammatory drug (NSAID) users. Sixty-nine percent had duodenal, 24% gastric, and 7% pyloric ulcer. Recurrence of bleeding was demonstrated in two patients at 1 year (incidence: 0.22% per patient-year of follow up), which occurred after NSAID use in both cases. CONCLUSION: Peptic ulcer rebleeding does not occur in patients with complicated ulcers after H. pylori eradication. Maintenance anti-ulcer (antisecretory) therapy is not necessary if eradication is achieved.     

Distribution and seasonal variability in the benthic eukaryotic community of Río Tinto (SW, Spain), an acidic, high metal extreme environment

The eukaryotic community of the Río Tinto (SW, Spain) was surveyed in fall, winter and spring through the combined use of traditional microscopy and molecular approaches, including Denaturing Gradient Gel Electrophoresis (DGGE) and sequence analysis of 18S rRNA gene fragments. Eukaryotic assemblages of surface sediment biofilms collected in January, May and September 2002 were compared from 13 sampling stations along the river. Physicochemical data revealed extremely acidic conditions (the pH ranged from 0.9 to 2.5) with high concentrations of heavy metals, including up to 20 mg l(-1) Fe, 317 mg l(-1) Zn, 47 mg l(-1) As, 42 mg l(-1) Cd and 4 mg l(-1) Ni. In total, 20 taxa were identified, including members of the Bacillariophyta, Chlorophyta and Euglenophyta phyla as well as ciliates, cercomonads, amoebae, stramenopiles, fungi, heliozoans and rotifers. In general, total cell abundances were highest in fall and spring but decreased drastically in winter, and the sampling stations with the most extreme conditions showed the lowest number of cells, as well as the lowest diversity. Species diversity did not vary much during the year. Only the filamentous algae showed a dramatic seasonal change, since they almost disappeared in winter and reached the highest biomass during the summer. Principal Components Analysis (PCA) showed a high inverse correlation between pH and most of the heavy metals analyzed, as well as Dunaliella sp., while Chlamydomonas sp. was directly related to pH during May and September. Three heavy metals (Zn, Cu and Ni) remained separate from the rest and showed an inverse correlation with most of the species analyzed, except for Dunaliella sp.