Stent thrombosis in patients treated with thienopyridines: clinical description of 10 cases

Subacute stent thrombosis (SAST) is a major thrombotic complication of coronary stenting. Its occurrence has been substantially reduced by thienopyridine treatment. However, information on clinical profile of patients with SAST in clopidogrel era is limited. In order to define the incidence and factors predisposing to stent thrombosis, we analyzed the computerized angiographic database of three interventional cardiology centers. Out of a total number of 5903 percutaneous coronary interventions (PCIs) with stent implantation, we found 10 patients with SAST (0.17%). The indication for PCI was usually an early invasive approach (90%) during an acute coronary syndrome. All patients were treated with an apparently optimal antithrombotic regimen (90% received heparin or LMWH and 70% received IIb/IIIa receptor inhibitors and all given aspirin). In each of the patients, we could identify high-risk angiographic findings. SAST presentation was always clinically significant with definite myocardial infarction in 100% of cases. 80% of cases occurred during the first six days post PCI. Two patients had a recurrent event. Finally, despite earlier reports of atorvastatin-mediated inhibition of clopidogrel activation we did not find any patient with SAST taking both drugs. Thus, patients with stent thrombosis during thienopyridine treatment usually exhibit high-risk angiographic features. Prospective studies should be performed to elucidate drug interactions that may reduce clopidogrel efficacy and contribute to stent thrombosis.     

The not-so-humble worm

A report of the Wellcome Trust meeting "Caenorhabditis elegans past, present and future: The not-so-humble worm", Hinxton, UK, 10 September 2003.     

Modulation of adenosine receptor affinity and intrinsic efficacy in adenine nucleosides substituted at the 2-position

We studied the structural determinants of binding affinity and efficacy of adenosine receptor (AR) agonists. Substituents at the 2-position of adenosine were combined with N(6)-substitutions known to enhance human A(3)AR affinity. Selectivity of binding of the analogues and their functional effects on cAMP production were studied using recombinant human A(1), A(2A), A(2B), and A(3)ARs. Mainly sterically small substituents at the 2-position modulated both the affinity and intrinsic efficacy at all subtypes. The 2-cyano group decreased hA(3)AR affinity and efficacy in the cases of N(6)-(3-iodobenzyl) and N(6)-(trans-2-phenyl-1-cyclopropyl), for which a full A(3)AR agonist was converted into a selective antagonist; the 2-cyano-N(6)-methyl analogue was a full A(3)AR agonist. The combination of N(6)-benzyl and various 2-substitutions (chloro, trifluoromethyl, and cyano) resulted in reduced efficacy at the A(1)AR. The environment surrounding the 2-position within the putative A(3)AR binding site was explored using rhodopsin-based homology modeling and ligand docking.     

Fluorescent labeling of endothelial cells allows in vivo,continuous characterization of the vascular development of Xenopus laevis

Appropriate blood supply and vascular development are necessary in development and in cancer, heart disease, and diabetes. Here, we report the use of DiI-labeled acetylated low-density lipoprotein (DiI-Ac-LDL) to label endothelial cells and characterize the vasculature of live Xenopus embryos. The atlas we have created provides a detailed map of normal vascular development against which perturbations of normal patterning can be compared. By following the development of the intersomitic vessels in real-time, we show that, while rostrocaudal gradient of maturing intersomitic vessels occurs, it is not absolute. In addition, the comparative study of the ontogeny of nerve bundles from the spinal cord of transgenic Xenopus embryos expressing green fluorescent protein in the nervous system and blood vessels demonstrates a strong anatomical correlation in neurovascular development. These studies provide the basis for understanding how the vascular system forms and assumes its complicated stereotypical pattern in normal development and in disease.     

Escherichia coli cells with increased levels of DnaA and deficient in recombinational repair have decreased viability

The dnaA operon of Escherichia coli contains the genes dnaA, dnaN, and recF encoding DnaA, beta clamp of DNA polymerase III holoenzyme, and RecF. When the DnaA concentration is raised, an increase in the number of DNA replication initiation events but a reduction in replication fork velocity occurs. Because DnaA is autoregulated, these results might be due to the inhibition of dnaN and recF expression. To test this, we examined the effects of increasing the intracellular concentrations of DnaA, beta clamp, and RecF, together and separately, on initiation, the rate of fork movement, and cell viability. The increased expression of one or more of the dnaA operon proteins had detrimental effects on the cell, except in the case of RecF expression. A shorter C period was not observed with increased expression of the beta clamp; in fact, many chromosomes did not complete replication in runout experiments. Increased expression of DnaA alone resulted in stalled replication forks, filamentation, and a decrease in viability. When the three proteins of the dnaA operon were simultaneously overexpressed, highly filamentous cells were observed (>50 micro m) with extremely low viability and, in runout experiments, most chromosomes had not completed replication. The possibility that recombinational repair was responsible for the survival of cells overexpressing DnaA was tested by using mutants in different recombinational repair pathways. The absence of RecA, RecB, RecC, or the proteins in the RuvABC complex caused an additional approximately 100-fold drop in viability in cells with increased levels of DnaA, indicating a requirement for recombinational repair in these cells.     

Type III protein translocase: HrcN is a peripheral ATPase that is activated by oligomerization

Type III protein secretion (TTS) is catalyzed by translocases that span both membranes of Gram-negative bacteria. A hydrophilic TTS component homologous to F1/V1-ATPases is ubiquitous and essential for secretion. We show that hrcN encodes the putative TTS ATPase of Pseudomonas syringae pathovar phaseolicola and that HrcN is a peripheral protein that assembles in clusters at the membrane. A decahistidinyl HrcN derivative was overexpressed in Escherichia coli and purified to homogeneity in a folded state. Hydrodynamic analysis, cross-linking, and electron microscopy revealed four distinct HrcN forms: I, 48 kDa (monomer); II, approximately 300 kDa (putative hexamer); III, 575 kDa (dodecamer); and IV, approximately 3.5 MDa. Form III is the predominant form of HrcN at the membrane, and its ATPase activity is dramatically stimulated (>700-fold) over the basal activity of Form I. We propose that TTS ATPases catalyze protein translocation as activated homo-oligomers at the plasma membrane.     

Can sucrose affect polymerase chain reaction product formation?

Sucrose or trehalose at 0.3 M stabilised double-stranded DNA in PCR. Furthermore, the DNA polymerase from Thermus aquaticus was stabilised towards thermo-denaturation at 98°C by sucrose or trehalose. The effect was limited to these sugars and was shown not to be general for carbohydrates.     

Improving coiled-coil stability by optimizing ionic interactions

Alpha-helical coiled coils are a common protein oligomerization motif stabilized mainly by hydrophobic interactions occurring along the coiled-coil interface. We have recently designed and solved the structure of a two-heptad repeat coiled-coil peptide that is stabilized further by a complex network of inter- and intrahelical salt-bridges in addition to the hydrophobic interactions. Here, we extend and improve the de novo design of this two heptad-repeat peptide by four newly designed peptides characterized by different types of ionic interactions. The contribution of these different types of ionic interactions to coiled-coil stability are analyzed by CD spectroscopy and analytical ultracentrifugation. We show that all peptides are highly alpha-helical and two of them are 100% dimeric under physiological conditions. Furthermore, we have solved the X-ray structure of the most stable of these peptides and the rational design principles are verified by comparing this structure to the structure of the parent peptide. We show that by combining the most favorable inter- and intrahelical salt-bridge arrangements it is possible to design coiled-coil oligomerization domains with improved stability properties.     

Delayed striate cortical activation during spatial attention

Recordings of event-related potentials (ERPs) and event-related magnetic fields (ERMFs) were combined with functional magnetic resonance imaging (fMRI) to study visual cortical activity in humans during spatial attention. While subjects attended selectively to stimulus arrays in one visual field, fMRI revealed stimulus-related activations in the contralateral primary visual cortex and in multiple extrastriate areas. ERP and ERMF recordings showed that attention did not affect the initial evoked response at 60-90 ms poststimulus that was localized to primary cortex, but a similarly localized late response at 140-250 ms was enhanced to attended stimuli. These findings provide evidence that the primary visual cortex participates in the selective processing of attended stimuli by means of delayed feedback from higher visual-cortical areas.     

Hedgehog-Gli signalling and the growth of the brain

The development of the vertebrate brain involves the creation of many cell types in precise locations and at precise times, followed by the formation of functional connections. To generate its cells in the correct numbers, the brain has to produce many precursors during a limited period. How this is achieved remains unclear, although several cytokines have been implicated in the proliferation of neural precursors. Understanding this process will provide profound insights, not only into the formation of the mammalian brain during ontogeny, but also into brain evolution. Here we review the role of the Sonic hedgehog-Gli pathway in brain development. Specifically, we discuss the role of this pathway in the cerebellar and cerebral cortices, and address the implications of these findings for morphological plasticity. We also highlight future directions of research that could help to clarify the mechanisms and consequences of Sonic hedgehog signalling in the brain.