Molecular dynamics simulations of a fibrillogenic peptide derived from apolipoprotein C-II

The pathway to amyloid fibril formation in proteins involves specific structural changes leading to the combination of misfolded intermediates into oligomeric assemblies. Recent NMR studies showed the presence of “turns” in amyloid peptides, indicating that turn formation may play an important role in the nucleation of the intramolecular folding and possible assembly of amyloid. Fully solvated all-atom molecular dynamics simulations were used to study the structure and dynamics of the apolipoprotein C-II peptide 56 to 76, associated with the formation of amyloid fibrils. The peptide populated an ensemble of turn structures, stabilized by hydrogen bonds and hydrophobic interactions enabling the formation of a strong hydrophobic core which may provide the conditions required to initiate aggregation. Two competing mechanisms discussed in the literature were observed. This has implications in understanding the mechanism of amyloid formation in not only apoC-II and its fragments, but also in other amyloidogenic peptides.     

Understanding the changes in the circular dichroism of light harvesting complex II upon varying its pigment composition and organization

In this work we modeled the circular dichroism (CD) spectrum of LHCII, the main light harvesting antenna of photosystem II of higher plants. Excitonic calculations are performed for a monomeric subunit, taken from the crystal structure of trimeric LHCII from spinach [Liu, Z. F., Yan, H. C., Wang, K. B., Kuang, T. Y., Zhang, J. P., Gui, L. L., An, X. M., and Chang, W. R. (2004) Nature 428, 287-292]. All of the major features of the CD spectrum above 450 nm are satisfactorily reproduced, and possible orientations of the Chl and carotenoid transition dipole moments are identified. The obtained modeling parameters are used to simulate the CD spectra of two complexes with altered pigment composition: a mutant lacking Chls a 611-612 and a complex lacking the carotenoid neoxanthin. By removing the relevant pigment(s) from the structure, we are able to reproduce their spectra, which implies that the alteration does not disturb the overall structure. The CD spectrum of trimeric LHCII shows a reversed relative intensity of the two negative bands around 470 and 490 nm as compared to monomeric LHCII. The simulations reproduce this reversal, indicating that it is mainly due to interactions between chromophores in different monomeric subunits, and the trimerization does not induce observable changes in the monomeric structure. Our simulated spectrum resembles one of two different trimeric CD spectra reported in literature. We argue that the differences in the experimental trimeric CD spectra are caused by changes in the strength of the monomer-monomer interactions due to the differences in detergents used for the purification of the complexes.     

Effects of PKA phosphorylation of cardiac troponin I and strong crossbridge on conformational transitions of the N-domain of cardiac troponin C in regulated thin filaments

Regulation of cardiac muscle function is initiated by binding of Ca2+ to troponin C (cTnC) which induces a series of structural changes in cTnC and other thin filament proteins. These structural changes are further modulated by crossbridge formation and fine-tuned by phosphorylation of cTnI. The objective of the present study is to use a new F?rster resonance energy transfer-based structural marker to distinguish structural and kinetic effects of Ca2+ binding, crossbridge interaction, and protein kinase A phosphorylation of cTnI on the conformational changes of the cTnC N-domain. The FRET-based structural marker was generated by attaching AEDANS to one cysteine of a double-cysteine mutant cTnC(13C/51C) as a FRET donor and attaching DDPM to the other cysteine as the acceptor. The doubly labeled cTnC mutant was reconstituted into the thin filament by adding cTnI, cTnT, tropomyosin, and actin. Changes in the distance between Cys13 and Cys51 induced by Ca2+ binding/dissociation were determined by FRET-sensed Ca2+ titration and stopped-flow studies, and time-resolved fluorescence measurements. The results showed that the presence of both Ca2+ and strong binding of myosin head to actin was required to achieve a fully open structure of the cTnC N-domain in regulated thin filaments. Equilibrium and stopped-flow studies suggested that strongly bound myosin head significantly increased the Ca2+ sensitivity and changed the kinetics of the structural transition of the cTnC N-domain. PKA phosphorylation of cTnI impacted the Ca2+ sensitivity and kinetics of the structural transition of the cTnC N-domain but showed no global structural effect on cTnC opening. These results provide an insight into the modulation mechanism of strong crossbridge and cTnI phosphorylation in cardiac thin filament activation/relaxation processes.     

Misexpression screen in Drosophila melanogaster aiming to reveal novel factors involved in formation of body parts

To identify novel factors that lead a fly imaginal disc to adopt its developmental fate, we carried out a modular dominant misexpression screen in imaginal discs. We have identified two factors that appear to change the fate of the respective body structure and appear to lead to the transformation of a body part. In one mutant line, notum tissue, normally derived from wing imaginal tissue, formed close to the site of the sternopleural bristles, which are leg disc derivatives. In the other line, the arista is transformed into a tubular structure, resembling an abnormal leg. We found that ectopic expression of abrupt was responsible for this potential transformation of the arista.     

Experimental manipulation of timing of breeding suggests laying order instead of breeding synchrony affects extra-pair paternity in house sparrows

The timing of breeding may not only affect breeding patterns such as the overlap of chick rearing period with the peak in food availability but also the opportunity for extra-pair mating. A negative relationship has been predicted between extra-pair paternity and breeding synchrony, assuming that male extra-pair activity is traded against mate guarding and parenting duties. In contrast, if female ability to assess male quality is temporally constrained, sperm competition might be a positive function of breeding synchrony. Here we manipulated the progress of nesting by nest material exchange within nesting aggregations to see whether the timing of breeding affects extra-pair paternity in house sparrows. We found that late broods within nesting clusters contained extra-pair young more often than early broods, but breeding synchrony did not turn out to be a significant predictor of extra-pair paternity. Our study indicates that temporal constraints of male extra-pair activity may account for extra-pair paternity levels, but it is also possible that late-breeding females may accept extra-pair copulations to ensure egg fertilization.     

Cervical screening in the UK and laboratory quality control in the context of the 2007 European guidelines

The first part of this article will provide an overview of cervical cancer screening in the UK during the years before, during and after the introduction of a highly successful centrally organised cervical screening programme in 1988: since then the incidence of invasive cervical cancer has fallen by more than 40%. Screening was introduced in a background of opportunistic screening with poor quality control during a period of time when risk of disease was increasing, which will be demonstrated by national registrations of carcinoma in situ as well as invasive cancer. The programme is still facing new challenges and has recently recorded falling screening coverage in younger women, the causes of which have yet to be established. Liquid-based cytology is in the process of being rolled out nationally but high-risk human papillomavirus testing has yet to be introduced into the National Health Service (NHS) programme. Lessons from our experience may be relevant to countries introducing and maintaining organised programmes elsewhere under similar circumstances. The second part of the article will consider laboratory quality control as practiced in the UK and as recommended in the second edition of the European Guidelines for Quality Assurance in Cervical Cancer Screening. These evidence-based guidelines provide recommendations for organising and monitoring quality control as well as for introducing new technology and standardising terminology, which are equally relevant for new and existing programmes. Invasive cancer audit may highlight areas where procedures could be improved in any programme but also can also demonstrate the effectiveness of screening.     

Preemptive heme oxygenase-1 gene delivery reveals reduced mortality and preservation of left ventricular function 1 yr after acute myocardial infarction

We reported previously that predelivery of heme oxygenase-1 (HO-1) gene to the heart by adeno-associated virus-2 (AAV-2) markedly reduces ischemia and reperfusion (I/R)-induced myocardial injury. However, the effect of preemptive HO-1 gene delivery on long-term survival and prevention of postinfarction heart failure has not been determined. We assessed the effect of HO-1 gene delivery on long-term survival, myocardial function, and left ventricular (LV) remodeling 1 yr after myocardial infarction (MI) using echocardiographic imaging, pressure-volume (PV) analysis, and histomorphometric approaches. Two groups of Lewis rats were injected with 2 x 10(11) particles of AAV-LacZ (control) or AAV-human HO-1 (hHO-1) in the anterior-posterior apical region of the LV wall. Six weeks after gene transfer, animals were subjected to 30 min of ischemia by ligation of the left anterior descending artery followed by reperfusion. Echocardiographic measurements and PV analysis of LV function were obtained at 2 wk and 12 mo after I/R. One year after acute MI, mortality was markedly reduced in the HO-1-treated animals compared with the LacZ-treated animals. PV analysis demonstrated significantly enhanced LV developed pressure, elevated maximal dP/dt, and lower end-diastolic volume in the HO-1 animals compared with the LacZ animals. Echocardiography showed a larger apical anterior-to-posterior wall ratio in HO-1 animals compared with LacZ animals. Morphometric analysis revealed extensive myocardial scarring and fibrosis in the infarcted LV area of LacZ animals, which was reduced by 62% in HO-1 animals. These results suggest that preemptive HO-1 gene delivery may be useful as a therapeutic strategy to reduce post-MI LV remodeling and heart failure.     

Induction of matrix metalloproteinase-2 enhances systemic arterial contraction after hypoxia

This study was carried out to determine the role of increased vascular matrix metalloproteinase-2 (MMP-2) expression in the changes in systemic arterial contraction after prolonged hypoxia. Rats and mice were exposed to hypoxia (10% and 8% O(2), respectively) or normoxia (21% O(2)) for 16 h, 48 h, or 7 days. Aortae and mesenteric arteries were either mounted in organ bath myographs or frozen in liquid nitrogen. MMP-2 inhibition with cyclic CTTHWGFTLC (CTT) reduced contraction to phenylephrine (PE) in aortae and mesenteric arteries from rats exposed to hypoxia for 7 days but not in vessels from normoxic rats. Similarly, CTT reduced contraction to Big endothelin-1 (Big ET-1) in aortae from rats exposed to hypoxia for 7 days. Responses to PE were reduced in hypoxic MMP-2(-/-) mice compared with MMP-2(+/+) mice. Increased contraction to Big ET-1 after hypoxia was observed in MMP-2(+/+) mice but not in MMP-2(-/-) mice. Rat aortic MMP-2 and membrane type 1 (MT1)-MMP protein levels and MMP activity were increased after 7 days of hypoxia. Rat aortic MMP-2 and MT1-MMP mRNA levels were increased in the deep medial vascular smooth muscle. We conclude that hypoxic induction of MMP-2 expression potentiates contraction in systemic conduit and resistance arteries. This may preserve the capacity to regulate the systemic circulation in the transition between the alterations in vascular tone and structural remodeling that occurs during prolonged hypoxic epochs.     

3,4-Dihydro-2H-benzoxazinones as dual-acting 5-HT1A receptor antagonists and serotonin reuptake inhibitors

Investigation of halogen substitution in lead compound 1 has led to the identification of analogues which combine high affinity for 5-HT(1A) receptors and potent serotonin reuptake inhibitory activity. Several compounds show an improved selectivity over 5-HT(1B) and 5-HT(1D) receptors and a superior pharmacokinetic profile in the rat.