Atomic structure and hierarchical assembly of a cross-β amyloid fibril

The cross-β amyloid form of peptides and proteins represents an archetypal and widely accessible structure consisting of ordered arrays of β-sheet filaments. These complex aggregates have remarkable chemical and physical properties, and the conversion of normally soluble functional forms of proteins into amyloid structures is linked to many debilitating human diseases, including several common forms of age-related dementia. Despite their importance, however, cross-β amyloid fibrils have proved to be recalcitrant to detailed structural analysis. By combining structural constraints from a series of experimental techniques spanning five orders of magnitude in length scale—including magic angle spinning nuclear magnetic resonance spectroscopy, X-ray fiber diffraction, cryoelectron microscopy, scanning transmission electron microscopy, and atomic force microscopy—we report the atomic-resolution (0.5 Å) structures of three amyloid polymorphs formed by an 11-residue peptide. These structures reveal the details of the packing interactions by which the constituent β-strands are assembled hierarchically into protofilaments, filaments, and mature fibrils.It is well established that a wide variety of peptides or proteins without any evident sequence similarity can self-assemble into amyloid fibrils (1, 2). These structures have many common characteristics, typically being 100–200 Å in diameter and containing a universal “cross-β” core structure composed of arrays of β-sheets running parallel to the long axis of the fibrils (3). These fibrillar states are highly ordered, with persistence lengths of the order of microns (4) and mechanical properties comparable to those of steel and dragline silk, and much greater than those typical of biological filaments such as actin and microtubules (5). Amyloid fibrils can also possess very high kinetic and thermodynamic stabilities, often exceeding those of the functional folded states of proteins (6), as well as a greater resistance to degradation by chemical or biological means (7). Several functional forms of proteins that exploit these properties have been observed in biological systems (8). More generally, however, the conversion of normally soluble functional proteins into the amyloid state is associated with many debilitating human disorders, ranging from Alzheimer’s disease to type II diabetes (1, 9). Our understanding of the nature of this type of filamentous aggregate has greatly improved in recent years (3, 10–19), particularly through the structural determination of their elementary β-strand building blocks (20) and the characterization of their assembly into cross-β steric zippers (21, 22). However, a thorough understanding of the hierarchical assembly of these individual structural elements into fully-formed fibrils, which display polymorphism but possess a range of generic features (23), has so far been limited by the absence of a complete atomic-resolution cross-β amyloid structures (2).We report here the simultaneous determination of the atomic-resolution structures of a cross-β amyloid fibril and two polymorphic variants, formed by an 11-residue fragment of the protein transthyretin, TTR(105–115) (20). These fibrils have the classic amyloid morphology, being 100–200 Å in diameter and typically 1–3 µm in length (SI Appendix, Fig. S1). We have achieved this objective by bringing together a set of complementary biophysical techniques to provide atomic structures of these complex aggregates. Specifically, we have combined interatomic structural restraints from magic angle spinning (MAS) nuclear magnetic resonance (NMR) spectroscopy with high-resolution electron density maps from cryoelectron microscopy (cryo-EM), together with data from X-ray fiber diffraction, scanning transmission electron microscopy (STEM), and atomic force microscopy (AFM) measurements. Our results reveal the molecular basis of the stability and polymorphism of these amyloid fibrils by defining at high resolution the variety of structural elements in their hierarchical self-assembly.     

Cardioversion for atrial fibrillation – how to prevent thromboembolic complications?

Abstract

Cardioversion is an essential component of rhythm control strategy for atrial fibrillation. The thromboembolic risk of cardioversion is well established and the mechanisms behind the phenomenon have been comprehensively described. There are several clinical aspects that are important to take into consideration when assessing the safety of cardioversion. Before proceeding to cardioversion, the probability of early treatment failure and antiarrhythmic treatment options to prevent recurrences should be carefully evaluated to avoid the risks of repeated futile cardioversions. Effective periprocedural anticoagulation is the mainstay in thromboembolic complication prevention and the first week after rhythm conversion is the most vulnerable period in this respect. Early timing of cardioversion appears to be another important measure to decrease the risk of thromboembolic complications. Transoesophageal echocardiography is useful in clinical scenarios where early cardioversion is desirable due to debilitating clinical symptoms and a short duration of arrhythmia or the adequacy of preceding anticoagulation is uncertain. However, it does not lessen the need for effective anticoagulation after cardioversion. This review summarizes the recent scientific discoveries to improve the safety of cardioversion for atrial fibrillation.

• Key messages

• Cardioversion for atrial fibrillation entails a significant risk of thromboembolic complications, especially during the first week after the procedure.

• The intensity of periprocedural anticoagulation and the timing of cardioversion appear to be significant determinants of the risk of thromboembolism.

• Awareness of the clinical aspects influencing cardioversion safety should be raised.

     

Effects of Postmenopausal Hormone Replacement Therapy With and Without Vitamin D3 on Circulating Levels of 25-Hydroxyvitamin D and 1,25-Dihydroxyvitamin D

The effects of postmenopausal hormone replacement therapy (HRT) and vitamin D₃ on vitamin D metabolites (25OHD and 1,25(OH)₂D) were studied in a population-based prospective 1-year study. The serum concentrations of intact parathyroid hormone (PTH), calcium, and phosphate were also studied. A total of 72 women were randomized into four treatment groups: HRT group (sequential combination of 2 mg estradiol valerate and 1 mg cyproterone acetate), Vit D₃ group (vitamin D₃ 300 IU/day + calcium lactate 500 mg/day), HRT + Vit D₃ group (both above) and placebo group (calcium lactate 500 mg/day). Serum samples were taken in March–April, when vitamin D formation from sunlight in Finland is minimal after the dark winter. Serum concentrations of 25OHD increased in the Vit D₃ group (33.5%, P < 0.001) and in the HRT + Vit D₃ group (38.2%, P < 0.001) but had not changed significantly in the HRT and placebo groups at the 1-year follow-up examination. Serum concentrations of calcitriol (1,25(OH)₂D) increased, however, only in the HRT group (23.7%, P < 0.05), and remained unchanged in other groups. Serum concentrations of PTH decreased by 23.2% (P < 0.05) in the placebo group, but did not change significantly in the other three groups. The concentrations of serum calcium increased in the nonhormone groups (P < 0.001), whereas serum phosphate concentrations decreased in the hormone groups (P < 0.05 and 0.001). Our results confirm the positive effect of 1 year of HRT on serum calcitriol. Vitamin D₃ supplementation increased 25OHD concentrations, but did not affect calcitriol concentrations even though the initial levels were low. Interestingly, the combination of HRT and vitamin D₃ did not increase serum calcitriol concentrations as much as HRT alone. Received: 14 June 1996 / Accepted: 17 June 1997     

Ruthenium red inhibits the voltage-dependent increase in cytosolic free calcium in cortical synaptosomes from guinea-pig

Effects of ruthenium red (RuR) on adenylates, plasma membrane potential (delta psi p) and cytosolic free calcium concentration ([Ca2+]c) in cortical synaptosomes from guinea-pig were investigated. Ten micromoles of RuR did not affect either energy levels as indicated by ATP/ADP ratio or the basal delta psi p. The resting [Ca2+]c in the presence of RuR was unchanged, but above 5 microM it inhibited by more than 50% of the voltage-activated increase in [Ca2+]c by K+-depolarization. In another experiment the potencies of 10 microM RuR and 100 microM verapamil to inhibit high K+-induced increase in [Ca2+]c were compared. It was found that either produced 59% inhibition and this inhibition was not potentiated by the substances together (65% inhibition). The extent of depolarisation of delta psi p by high external K+ was independent of the presence of RuR. RuR blocked only 20% of the increase in [Ca2+]c by veratridine treatment, indicating that Ca2+ accumulation into synaptosomal cytoplasm by veratridine involves some additional mechanisms other than depolarisation of delta psi p. The mechanism of inhibition of evoked release of neurotransmitters by RuR is discussed.     

Intracellular calcium as an index of neurotoxic damage

The availability of techniques that allow the quantitation of levels of ionized calcium within intact cells and synaptosomes, allows a new approach to understanding the events underlying neurotoxicity. By use of various pharmacological agents, it is possible to dissect out vulnerable loci within the cell that account for the increases in cytosolic calcium which accompany many neurotoxic events. The relation between calcium and cell death can now be more concisely addressed. This article gives examples of the types of questions that this new methodology can resolve.     

Röntgenologische Lungenbefunde beim ersten Atemzug des Neugeborenen

Zusammenfassung Das Geschehen des ersten Atemzuges wurde mit Kineröntgenfilm bei 17 und mit Filmwechsler bei 34 Neugeborenen untersucht. Dabei zeigte sich, daß die primäre Aeration gewöhnlich unten in der linken Lunge anfängt. Vermutlich beruht das auf der größeren Beweglichkeit der linken Zwerchfellhälfte. Zwischen Geburt und Aeration liegt ein Intervall von verschieden langer Dauer. Wenn die Luft aber in Bewegung gekommen ist, so verbreitet sie sich explosionsartig über die Lungenfelder. Die Aeration scheint vor allem durch eine starke Kontraktion des Zwerchfells bewirkt zu sein, obwohl noch weitere, bisher weniger bekannte Umstände, wie Veränderungen im Lungenblutkreislauf, daran beteiligt sein dürften.     

Release and inhibition of uptake of 5-hydroxytryptamine in blood platelets in vitro by copper and methyl mercury

Divalent copper (Cu) and methyl mercury (Met-Hg) are potent inhibitors of the uptake of 5-hydroxytryptamine (5-HT) in rat hypothalamic synaptosomes in vitro. To assess the usefulness of blood platelets as a peripheral model of central serotonergic nerve endings for neurotoxicological studies, for comparison human and rabbit platelets were utilized. Cu inhibited 5-HT uptake into human platelets when platelets were separated from plasma (the IC50 0.7-0.8 microM). Plasma added with platelets abolished the toxic influence of Cu towards platelets. Met-Hg inhibited 5-HT uptake both in washed platelets (the IC50 0.2 microM) and in platelets added in plasma (the IC50s 10-15 microM). The inhibition of uptake by Met-Hg did not depend on buffer Ca and Mg. At low concentrations of Cu, the uptake tended to be more inhibited in the presence of Ca and Mg. Zn and Hg did not affect 5-HT uptake up to 100 microM. Pb inhibited it transiently (28% at 1 microM) in the presence of Ca and Mg. Met-Hg induced the release of endogenous 5-HT from rabbit platelets when they were in a suspension in Ca-free buffer, but Cu did not, even at 100 microM concentration. The results suggest firstly, that blood platelets give results comparable with brain synaptosomes regarding inhibitory effects of metals on 5-HT uptake provided plasma is decanted. Secondly, the inhibition of 5-HT uptake by Cu appears to be purely plasma membrane related but Met-Hg may, in addition, induce release of 5-HT from storage granules.     

Untersuchungen über die in vitro zur Entfaltung von Neugeborenenlungen nötigen Druckwerte

Zusammenfassung Mit der in Abb. 1 dargestellten Versuchsanordnung wurde der zur Entfaltung fetaler Meerschweinchenlungen benötigte Druck bestimmt. Es ist gleichgültig, ob man die Druckdifferenz durch trachealen Überdruck oder durch Unterdruck im Thorax erzeugt (Durchschnittswert 16 mm Hg; Abb. 2).Die Unterbindung des Lungenkreislaufs erhöht den benötigten Druck um 30–50% (Abb. 3). Diese Beobachtung unterstützt die Auffassung, daß der Lungenkreislauf eine wesentliche Rolle beim Belüftungsvorgang der Neugeborenenlungen spielt. Die histologischen Schnitte der expandierten Lungen zeigten, daß die Belüftung in den peripheren Gebieten der Lungen beginnt.