Binding of 9-aminoacridine to deoxydinucleoside phosphates of defined sequence: Preferences and stereochemistry

The effect of sequence on the binding of 9-aminoacridine to DNA has been investigated by studying its interaction with deoxydinucleoside phosphates of different sequences using proton nuclear magnetic resonance. Quantitative binding information can be obtained by comparison of the proton chemical shift behavior of 9-aminoacridine upon addition of dinucleoside phosphate to various models for the interaction using least-squares computer fitting procedures. The simplest model that fits the data includes (1) dimerization of 9-aminoacridine and (2) a mixture of 1:1 and 2:1 (dinucleoside phosphate/9-aminoacridine) complexes. The computed parameters allow comparison of binding constants and stereochemistry for different sequences. The 1:1 complexes seem to involve interaction of the ring nitrogen with the backbone phosphate and stacking of one or both chromophores on the acridine; preference in binding is observed for alternating (purine-pyrimidine or pyrimidine-purine) over non-alternating (purine-purine) dinucleoside phosphates. The 2:1 complexes involve intercalation of the acridine between two complementary dinucleoside phosphate strands with weak sequence preferences in binding. The stereochemistry of intercalation differs between non-alternating purine-purine sequences and the alternating pyrimidine-purine or purine-pyrimidine sequences in having the 9-aminoacridine stacked with the purines of one strand rather than straddling the purines on opposite strands. The difference in stereochemistry could possibly be a determining factor in frameshift sequence specificity.     

Globular Tetramers of β2-Microglobulin Assemble into Elaborate Amyloid Fibrils

Amyloid fibrils are ordered polymers in which constituent polypeptides adopt a non-native fold. Despite their importance in degenerative human diseases, the overall structure of amyloid fibrils remains unknown. High-resolution studies of model peptide assemblies have identified residues forming cross-β-strands and have revealed some details of local β-strand packing. However, little is known about the assembly contacts that define the fibril architecture. Here we present a set of three-dimensional structures of amyloid fibrils formed from full-length β₂-microglobulin, a 99-residue protein involved in clinical amyloidosis. Our cryo-electron microscopy maps reveal a hierarchical fibril structure built from tetrameric units of globular density, with at least three different subunit interfaces in this homopolymeric assembly. These findings suggest a more complex superstructure for amyloid than hitherto suspected and prompt a re-evaluation of the defining features of the amyloid fold.     

The restrictive proteolysis of α-fodrin to a 120 kDa fragment is not catalyzed by calpains during thymic apoptosis

The -subunit (240 kDa) of fodrin was found to be digested selectively to a 120 kDa fragment during apoptosis of rat thymocytes in vivo and in vitro. This fragment was detected by an antibody (Ab) against full length -fodrin, but not by the anti-N-terminal sequence (GMMPR) of the -calpain-generated 150 kDa fragment Ab or the anti-PEST sequence of -fodrin Ab. On the other hand, basal levels of the 150 kDa fragment were constantly recognized by these three antibodies during apoptosis. The production of the 120 kDa fragment during apoptosis was not affected by the addition of calpain inhibitors such as Ac-LLLnal and E-64d, despite inhibition of the generation of the 150 kDa fragment. When x-irradiated thymocytes were incubated in the presence of N-tosyl-L-phenylalanyl chloromethyl ketone (TPCK), both production of the 120 kDa fragment and apoptosis were suppressed. Purified - and m-calpain did not catalyze the formation of the 120 kDa fragment from purified -fodrin in vitro. These results suggest that a protease different from calpains is involved in the major process of -fodrin proteolysis to a 120 kDa fragment during thymic apoptosis.     

Investigation of 0.2 µm filterable bacteria from the Western Mediterranean Sea using a molecular approach: dominance of potential starvation forms

Although the existence of 0.2 μm filterable bacteria has been known since the early 80's, they are not taken into consideration when modeling microbial food webs, due to an overall lack of information concerning this specific size class. According to physiological studies on starvation forms and investigations on small bacterial cells in marine ecosystems, a 0.2 μm filtrate may consist of different phenotypes: starvation forms of typical marine bacteria, ultramicrobacteria or bacterial cells, even larger than 0.2 μm, but flexible enough to pass the nominal filter pore-size. In this pilot study we examined three filtered seawater fractions from the Western Mediterranean Sea (Bay of Calvi, Corsica/France) - the total bacterial population, the bacterial fraction above 0.2 μm and the 0.2 μm filtrate - to investigate the bacterial community structure of each of those fractions by the molecular approach of denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments. The analysis of the resulting DGGE profiles revealed different patterns of dominant bands for the 0.2 μm filterable and the total bacterial populations within the samples. Additionally the 0.2 μm filterable bacterial compartment exhibited obvious differences in band patterns for winter and summer samples, which were not observed for the total bacterial fraction. According to the current knowledge concerning the status of 0.2 μm filterable bacteria, DGGE patterns indicate that most of the fragments representing 0.2 μm filterable bacteria were rather starvation forms of marine bacteria than ultramicrobacteria. The sequencing of excised and cloned DNA bands of the DGGE profiles characterized the phylogenetic affiliation of the corresponding 0.2 μm filterable bacteria, clustering mainly with known, typical marine isolates of both alpha-subclass and gamma-subclass of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides branch.     

Δ5 Desaturase activity in rat kidney microsomes

Rat kidney microsomal fraction is able to catalyze the enzymatic desaturation of eicosatrienoic acid (20:3n-6) to arachidonic acid (20:4n-6) by the 5 desaturase pathway, in the presence of reduced nicotinamide adenine dinucleotide (NADH), adenosinetriphosphate (ATP) and coenzyme A (CoA). The substrate of the reaction [1-¹⁴C]eicosa-8,11,14trienoic acid (20:3n-6), was separated from the product [1-¹⁴C]eicosa-5,8,11,14-tetraenoic acid (20:4n-6) by reverse phase high-pressure liquid chromatography (RP-HPLC). These fatty acids were individually collected by monitoring the eluent at 205 nm and their radioactivity was measured by liquid scintillation counting. The 5 desaturase activity in kidney microsomes increased linearly with the substrate concentration up to 20 M. Enzymatic activity was sensitive to pH with the maximum at 7.0 and was proportional with incubation time up to 10 min. The apparent Km and Vmax of 5 desaturase were 56 M and 60 pmoles·min^–1·mg^–1 microsomal protein, respectively. Neither the cytosolic renal fraction nor the cytosolic liver fraction enhanced the 5 desaturase activity. Contrary to a report but in accordance to others, the present results suggest that rat kidneys can synthesize arachidonic acid at least to satisfy partially their needs for eicosanoid production.     

Non-fluoroscopic Catheter Tracking for Fluoroscopy Reduction in Interventional Electrophysiology

A technological platform (MediGuide) has been recently introduced for non-fluoroscopic catheter tracking. In several studies, we have demonstrated that the application of this non-fluoroscopic catheter visualization system (NFCV) reduces fluoroscopy time and dose by 90-95% in a variety of electrophysiology (EP) procedures. This can be of relevance not only to the patients, but also to the nurses and physicians working in the EP lab. Furthermore, in a subset of indications such as supraventricular tachycardias, NFCV enables a fully non-fluoroscopic procedure and allows the lab staff to work without wearing lead aprons. With this protocol, we demonstrate that even complex procedures such as ablations of atrial fibrillation, that are typically associated with fluoroscopy times of >30 min in conventional settings, can safely be performed with a reduction of >90% in fluoroscopy exposure by the additional use of NFCV.     

Activation of SIRT1 by curcumin blocks the neurotoxicity of amyloid-β25–35 in rat cortical neurons

As one of the most important hallmarks of Alzheimer’s disease (AD), β-amyloid (Aβ) plays important roles in inducing reactive oxygen species (ROS) generation, mitochondrial dysfunction and apoptotic cell death in neurons. Curcumin extracted from the yellow pigments spice plant turmeric shows multiplied bioactivities such as antioxidant and anti-apoptosis properties in vitro and in vivo. In the present study, the neuroprotective effect of curcumin against Aβ_25–35-induced cell death in cultured cortical neurons was investigated. We found that pretreatment of curcumin prevented the cultured cortical neurons from Aβ_25–35-induced cell toxicity. In addition, curcumin improved mitochondrial membrane potential (ΔΨm), decreased ROS generation and inhibited apoptotic cell death in Aβ_25–35 treated neurons. Furthermore, we found that application of curcumin activated the expression of SIRT1 and subsequently decreased the expression of Bax in the presence of Aβ_25–35. The protective effect of curcumin was blocked by SIRT1 siRNA. Taken together, our results suggest that activation of SIRT1 is involved in the neuroprotective action of curcumin.     

Use of deuterium labelling from deuterium oxide to demonstrate carotenoid transformations in photosynthetic bacteria

Carotenoid biosynthesis in many purple photosynthetic bacteria of the Rhodospirillaceae is inhibited by nicotine, and biosynthetic intermediates accumulate. If the inhibitor is removed and the bacteria are then incubated in buffered 99.6% deuterium oxide, deuterium is incorporated specifically into the C-2 position in both cyclic and acyclic carotenoids that are then formed from the previously accumulated hydrocarbon precursors. The deuterated molecular species can be detected and assayed by mass spectrometry. By use of this procedure, direct proof has been obtained for the conversion of lycopene into β-carotene and rhodopin in Rhodomicrobium vannielii, of neurosporene into spheroidene in Rhodopseudomonas sphaeroides and of spheroidene into hydroxyspheroidene in Rps. gelatinosa. The results confirm the operation of the biosynthetic pathways postulated for these organisms, and prove that formation of the acyclic 1-hydroxy-1,2-dihydro end-group characteristic of the carotenoids of photosynthetic bacteria occurs by addition of water to the C-1,2 double band.