Biochemical characterization of a CDC6-like protein from the crenarchaeon Sulfolobus solfataricus

Cdc6 proteins play an essential role in the initiation of chromosomal DNA replication in Eukarya. Genes coding for putative homologs of Cdc6 have been also identified in the genomic sequence of Archaea, but the properties of the corresponding proteins have been poorly investigated so far. Herein, we report the biochemical characterization of one of the three putative Cdc6-like factors from the hyperthermophilic crenarchaeon Sulfolobus solfataricus (SsoCdc6-1). SsoCdc6-1 was overproduced in Escherichia coli as a His-tagged protein and purified to homogeneity. Gel filtration and glycerol gradient ultracentrifugation experiments indicated that this protein behaves as a monomer in solution (molecular mass of about 45 kDa). We demonstrated that SsoCdc6-1 binds single- and double-stranded DNA molecules by electrophoretic mobility shift assays. SsoCdc6-1 undergoes autophosphorylation in vitro and possesses a weak ATPase activity, whereas the protein with a mutation in the Walker A motif (Lys-59 --> Ala) is completely unable to hydrolyze ATP and does not autophosphorylate. We found that SsoCdc6-1 strongly inhibits the ATPase and DNA helicase activity of the S. solfataricus MCM protein. These findings provide the first in vitro biochemical evidence of a functional interaction between a MCM complex and a Cdc6 factor and have important implications for the understanding of the Cdc6 biological function.     

Differential effects of quercetin and resveratrol on Band 3 tyrosine phosphorylation signalling of red blood cells

The protective effects of eating fruits and vegetables in the prevention of several degenerative pathologies have been attributed at least in part to the antioxidant and anti-inflammatory properties of polyphenols. In this study, we investigated the effects of two polyphenols, quercetin and resveratrol, on red blood cell Band 3 tyrosine phosphorylation signalling activated by peroxynitrite. Peroxynitrite is a physiological oxidant scavenged largely by the erythrocyte and formed by the reaction between nitrogen monoxide and superoxide anion. Quercetin and its structurally analogous (+)-catechin inhibited the peroxynitrite-dependent upregulation of Band 3 tyrosine phosphorylation. Quercetin was found to downregulate the activity of syk, which is upstream in the Band 3 tyrosine phosphorylation cascade, and partially prevented peroxynitrite-mediated phosphotyrosine phosphatase inhibition. Resveratrol and hydroxytyrosol, unexpectedly, amplified peroxynitrite-dependent upregulation of Band 3 tyrosine phosphorylation through the activation of lyn, a kinase of the src family. The present results clearly indicate that polyphenols may activate cell transduction pathways in different and sometimes opposite ways.     

Increased acetylcholinesterase activities in specimens of Sparus auratus exposed to sublethal copper concentrations

The present study looks at possible changes in the activity of acetylcholinesterase (AChE) in tissues (brain and white muscle) of the Mediterranean bony fish Sparus auratus after a 20 days exposure to sublethal concentrations (0.1 or 0.5 ppm) of copper in the marine water and on control untreated animals. The trials also included measurements of Cu concentration in the tissues to evaluate possible metal accumulation. Moreover, sedimentation analysis as well as V(max) and K(m) determination were carried out in tissue extracts of Cu-exposed or control animals. V(max) and K(m) were also determined with or without addition of Cu(2+) in the assay. No Cu accumulation occurred in brain and muscle after Cu exposure. AChE showed in both tissues a molecular polymorphism with putative globular (G) and asymmetric (A) forms. Cu exposition led to an increased specific activity and improved catalytic efficiency of AChE in brain and muscle, seemingly regarding G forms. The increase in catalytic efficiency also resulted from the in vitro assay with tissue extracts and Cu(2+) addition. The higher AChE activity and catalytic efficiency in both tissues after Cu exposition and without metal accumulation, suggests an increase of free Cu aliquot into the cells, likely due to mechanisms of metal homeostasis.     

How well can blood pressure be controlled? Progress report on the Systolic Hypertension in Europe Follow-Up Study (Syst-Eur 2)

Background

The randomised, double-blind, placebo-controlled Systolic Hypertension in Europe trial (Syst-Eur 1) proved that blood pressure (BP) lowering therapy starting with nitrendipine reduces the risk of cardiovascular complications in elderly patients with isolated systolic hypertension. In an attempt to confirm the safety of long-term antihypertensive therapy based on a dihydropyridine, the Syst-Eur patients remained in open follow-up after the end of Syst-Eur 1. This paper presents the second progress report of this follow-up study (Syst-Eur 2). It describes BP control and adherence to study medications.

Methods

After the end of Syst-Eur 1 all patients, treated either actively or with placebo, were invited either to continue or to start antihypertensive treatment with the same drugs as previously used in the active treatment arm. In order to reach the target BP (sitting SBP <150 mmHg), the first line agent, nitrendipine, could be associated with enalapril and/or hydrochlorothiazide.

Results

Of the 3787 eligible patients, 3516 (93%) entered Syst-Eur 2. At the last available visit, 72% of the patients were taking nitrendipine. SBP/DBP at entry in Syst-Eur 2 averaged 160/83 mmHg in the former placebo group and 151/80 mmHg in the former active-treatment group. At the last follow-up visit SBP/DBP in the patients previously randomised to placebo or active treatment had decreased by 16/5 mmHg and 7/5 mmHg, respectively. The target BP was reached by 74% of the patients.

Conclusion

Substantial reductions in systolic BP may be achieved in older patients with isolated systolic hypertension with a treatment strategy starting with the dihydropyridine calcium-channel blocker, nitrendipine, with the possible addition of enalapril and/or hydrochlorothiazide.     

Rat carotid artery dilation by PTCA balloon catheter induces neointima formation in presence of IEL rupture

The best animal angioplasty model is the porcine model, which is expensive and not available in all laboratories. The aim of this study was to describe a new rat model of angioplasty. An injury was induced with the use of a standard percutaneous transluminal coronary angioplasty (PTCA) 1.5-mm balloon catheter. The neointimal tissue, arterial dimensions, and the injury index were assessed following angioplasty. Ki-67 expression was detected to evaluate cell turnover after balloon angioplasty. In contrast with the standard Clowes model, a significant neointimal formation was detected only in the presence of ruptured internal elastic lamina (IEL). A positive correlation between the percentage of ruptured IEL and the amount of neointimal tissue was also demonstrated. The percentage of IEL fracture correlates with the proliferation index by anti-Ki-67 immunolabeling 7 and 14 days after the angioplasty. Significant arterial negative remodeling was observed following PTCA balloon dilation. In conclusion, our inexpensive animal model of restenosis after angioplasty may have great relevance toward a better understanding of the mechanisms and toward assessment of new therapeutical strategies for this phenomenon.     

Morning preference in onset of symptomatic third-degree atrioventricular heart block

The aim of this study was to examine 24h patterning in the symptoms indicative of third-degree atrio-ventricular (AV) heart block. We found a total of 227 cases (126 men and 101 women) of third-degree AV block that had been diagnosed by the Emergency Medical Department of the St. Anna Hospital in Ferrara, Italy between 1990 and 2001. Determination of the hour of onset of symptomatic third-degree AV block, however, was possible and listed in the records of only 161 or 70.9% of the cases (92 men and 69 women). The onset time of every event was categorized into one of four 6h spans of the 24h: night (00:00-05:59h), morning (06:00-11:59h), afternoon (12:00-17:59h), and evening (18:00-23:59h). The onset of the symptoms of third-degree AV block in the sample of 161 cases was significantly greater in the morning between 06:00 and 11:59h than any other 6h span of the day and night (chi2-test; p < 0.001). The same phenomenon was substantiated in the subgroup of the 92 males (chi2; p < 0.0001), although it could not be detected for the smaller subgroup of 69 women. The 24h pattern, with morning preference, in the onset of symptomatic third-degree AV block is similar to the one in sudden cardiac death and cardiogenic cardiac arrest. The etiology of the 24h pattern in symptomatic AV block is unknown; it may be an expression of intrinsic biological rhythmicity within the heart tissue or its control system, and/or the timing of environmental triggers resulting in coronary ischemia.     

A genus-level supertree of the Dinosauria

One of the ultimate aims of systematics is the reconstruction of the tree of life. This is a huge undertaking that is inhibited by the existence of a computational limit to the inclusiveness of phylogenetic analyses. Supertree methods have been developed to overcome, or at least to go around this problem by combining smaller, partially overlapping cladograms. Here, we present a very inclusive generic-level supertree of Dinosauria (covering a total of 277 genera), which is remarkably well resolved and provides some clarity in many contentious areas of dinosaur systematics.     

Response to oxidative stress as a welfare parameter in swine

In pigs, the genetic selection for lean, large muscle blocks and fast growth has been linked to an increased prevalence of metabolic diseases such as porcine stress syndrome and mulberry heart disease. These diseases are associated with cardiovascular inadequacy, which may lead to oxidative stress. In the present study, reactive oxygen metabolites (ROMs) and the anti-oxidant power (OXY) in sera of different swine groups were investigated. The following groups were selected (each around 80 kg body weight): wild boars (WB), Cinta Senese (CS), and Landrace x Large White (LxLW), the latter as both specific pathogen-free (SPF) and intensively farmed animals. In addition, a group of LxLW agonic sows (AS) was also investigated; this group is known to be under oxidative stress. Two colorimetric micro-methods were used to measure ROMs and OXY; ROMs were expressed as mM H(2)O(2) and OXY as microM HOCl neutralised. Between groups, average ROM and OXY values were found to be significantly different by one-way ANOVA (P < 0.001). ROM levels were lower in WB (13.41 +/- 1.85) and CS (19.27 +/- 1.68), and highest in LxLW (42.00 +/- 1.36). OXY values ranged from 260.10 +/- 22.13 (WB) to 396.90 +/- 9.83 (LxLW). Only one swine group (the CS group) showed a significant, positive correlation between ROM and OXY values. The AS group even showed a negative correlation between ROM and OXY values. These results imply satisfactory environmental coping occurred only within the CS group. Results are discussed in the light of animal welfare legislation, food safety and consumers' protection.