Understanding pH-dependent selectivity of alamethicin K18 channels by computer simulation

Alamethicin K18 is a covalently linked alamethicin dimer in which the glutamine residue at position 18 in each helix has been replaced by a lysine residue. As described in previous work, channels formed by this peptide show pH-dependent selectivity. The maximum anion selectivity of the putative octameric conducting state is obtained at pH 7 or lower. Inasmuch as no change in selectivity is seen between pH 7 and pH 3, and because protons are expected to be in equilibrium with the open state of the channel during a selectivity measurement, the channel is believed to be fully charged (i.e., all eight lysines protonated) at pH 7. In an effort to understand how such a highly charged channel structure is stable in membranes and why it is not more selective for anions, we have performed a number of computer simulations of the system. Molecular dynamics simulations of 10 ns each of the octameric bundle in a lipid bilayer environment are presented, with either zero, four, or eight lysines charged in the absence of salt, and with eight lysines charged in the presence of 0.5 M and 1 M KCl. When no salt is present and all lysines are charged, on average 1.9 Cl(-) ions are inside the channel and the channel significantly deforms. With 0.5 M KCl present, 2.9 Cl(-) ions are inside the channel. With 1 M KCl present, four Cl(-) ions are present and the channel maintains a regular structure. Poisson-Boltzmann calculations on models of the octameric channel also predict an average of 2-4 Cl(-) ions near the lysine residues as a function of ionic strength. These counterions lower the apparent charge of the channel, which may underlie the decrease in selectivity observed experimentally with increasing salt concentrations. We suggest that to increase the selectivity of Alm K18 channels, positive charges could be engineered in a narrower part of the channel.     

Physiological adjustments to arid and mesic environments in larks (Alaudidae)

Because deserts are characterized by low food availability, high ambient temperature extremes, and absence of drinking water, one might expect that birds that live in these conditions exhibit a lower basal metabolic rate (BMR), reduced total evaporative water loss (TEWL), and greater ability to cope with high air temperatures than their mesic counterparts. To minimize confounding effects of phylogeny, we compared the physiological performance of four species of larks at ambient temperatures (T(a)'s) ranging from 0 degrees to 50 degrees C: hoopoe larks (Alaemon alaudipes) and Dunn's larks (Eremalauda dunni) live in hot and dry deserts, whereas skylarks (Alauda arvensis) and woodlarks (Lullula arborea) occur in temperate mesic areas. Mass-adjusted BMR and TEWL were indistinguishable between hoopoe lark and Dunn's lark and between skylark and woodlark. When grouping the data of the two desert larks in one set and the data of the two mesic larks in another, desert larks are shown to have 43% lower BMR levels and 27% lower TEWL values than the mesic species. Their body temperatures (T(b)'s) were 1.1 degrees C lower, and the minimal dry heat transfer coefficients (h) were 26% below values for the mesic larks. When T(a) exceeded T(b), the h of hoopoe larks and Dunn's larks was high and indistinguishable from h at 40 degrees C, in contrast to the prediction that h should be decreased to minimize heat gain through conductance, convection, or radiation from the environment when T(a) exceeds T(b).     

A case of spontaneous coronary artery disease regression

Coronary angiographic trials have demonstrated that lowering cholesterol can slow the progression of atherosclerosis, limit the formation of new lesions and enhance atherosclerotic regression together with reducing the incidence of clinical events (Waters D, 1996). Spontaneous regression of coronary atherosclerotic lesions is rare. We report the case of a patient with a severe within-stent restenotic lesion whose coronary disease spontaneously regressed 12 months after initial diagnosis, allowing for medical treatment of symptoms rather than repeated intervention. (Int J Cardiovasc Interventions 1999; 2: 121-123)     

Gene regulatory divergence among species estimated by altered developmental patterns in interspecific hybrids

Disturbances in the schedules of gene expression in developing interspecific fish hybrids have been used to draw inferences about the extent of gene regulatory divergence between species and about the degree to which this gene regulatory divergence is correlated with structural gene divergence, as estimated by genetic distance. Sperm from each of 10 different species representing six genera within the family Centrarchidae was used to fertilize eggs of the Florida largemouth bass (Micropterus salmoides floridanus). The genetic distances (D; Nei 1978) between the parental species used to form the hybrids ranged from 0.133 to 0.974. The developmental success and temporal patterns of gene expression of each of the hybrids were compared with those of the Florida largemouth bass. As the genetic distance between the paternal species and the Florida largemouth bass increased, there was a general decline in developmental success in the hybrid embryos as demonstrated by the observed reductions in the percentage of hatching and by progressively earlier and more extensive morphological abnormalities. Concomitantly, progressively more marked alterations in developmental schedules of expression of 15 enzyme loci occurred in the hybrids as the genetic distance between parental species increased. However, observed deviations from this trend for a few species may represent an uncoupling of the rates and modes of evolution of structural genes from those for genes regulating developmental processes.      

Nest survival in year‐round breeding tropical red‐capped larks Calandrella cinerea increases with higher nest abundance but decreases with higher invertebrate availability and rainfall

Nest survival is critical to breeding in birds and plays an important role in life‐history evolution and population dynamics. Studies evaluating the proximate factors involved in explaining nest survival and the resulting temporal patterns are biased in favor of temperate regions. Yet, such studies are especially pertinent to the tropics, where nest predation rates are typically high and environmental conditions often allow for year‐round breeding. To tease apart the effects of calendar month and year, population‐level breeding activity and environmental conditions, we studied nest survival over a 64‐month period in equatorial, year‐round breeding red‐capped larks Calandrella cinerea in Kenya. We show that daily nest survival rates varied with time, but not in a predictable seasonal fashion among months or consistently among years. We found negative influences of flying invertebrate biomass and rain on nest survival and higher survival of nests when nests were more abundant, which suggests that nest predation resulted from incidental predation. Although an increase in nest predation is often attributed to an increase in nest predators, we suggest that in our study, it may be caused by altered predator activity resulting from increased activity of the primary prey, invertebrates, rather than activity of the red‐capped larks. Our results emphasize the need to conduct more studies in Afro‐tropical regions because proximate mechanisms explaining nest predation can be different in the unpredictable and highly variable environments of the tropics compared with the relatively predictable seasonal changes found in temperate regions. Such studies will aid in better understanding of the environmental influences on life‐history variation and population dynamics in birds.     

Docking of mu-conotoxin GIIIA in the sodium channel outer vestibule

mu-Conotoxin GIIIA (mu-CTX) is a high-affinity ligand for the outer vestibule of selected isoforms of the voltage-gated Na(+) channel. The detailed bases for the toxin's high affinity binding and isoform selectivity are unclear. The outer vestibule is lined by four pore-forming (P) loops, each with an acidic residue near the mouth of the vestibule. mu-CTX has seven positively charged residues that may interact with these acidic P-loop residues. Using pair-wise alanine replacement of charged toxin and channel residues, in conjunction with double mutant cycle analysis, we determined coupling energies for specific interactions between each P-loop acidic residue and selected toxin residues to systematically establish quantitative restraints on the toxin orientation in the outer vestibule. Xenopus oocytes were injected with the mutant or native Na(+) channel mRNA, and currents measured by two-electrode voltage clamp. Mutant cycle analysis revealed novel, strong, toxin-channel interactions between K9/E403, K11/D1241, K11/D1532, and R19/D1532. Experimentally determined coupling energies for interacting residue pairs provided restraints for molecular dynamics simulations of mu-CTX docking. Our simulations suggest a refined orientation of the toxin in the pore, with toxin basic side-chains playing key roles in high-affinity binding. This modeling also provides a set of testable predictions for toxin-channel interactions, hitherto not described, that may contribute to high-affinity binding and channel isoform selectivity.     

No downregulation of immune function during breeding in two year‐round breeding bird species in an equatorial East African environment

Some equatorial environments exhibit substantial within‐location variation in environmental conditions throughout the year and yet have year‐round breeding birds. This implies that breeding in such systems are potentially unrelated to the variable environmental conditions. By breeding not being influenced by environmental conditions, we become sure that any differences in immune function between breeding and non‐breeding birds do not result from environmental variation, therefore allowing for exclusion of the confounding effect of variation in environmental conditions. This create a unique opportunity to test if immune function is down‐regulated during reproduction compared to non‐breeding periods. We compared the immune function of sympatric male and female chick‐feeding and non‐breeding red‐capped Calandrella cinerea and rufous‐naped larks Mirafra africana in equatorial East Africa. These closely‐related species occupy different niches and have different breeding strategies in the same grassland habitat. Red‐capped larks prefer areas with short grass or almost bare ground, and breed during low rainfall periods. Rufous‐naped larks prefer areas of tall grass and scattered shrubs and breed during high rainfall. We measured the following immune indices: nitric oxide, haptoglobin, agglutination and lysis, and measured total monthly rain, monthly average minimum (T_min) and maximum (T_max) temperatures. Contrary to our predictions, we found no down‐regulation of immune function during breeding; breeding birds had higher nitric oxide than non‐breeding ones in both species, while the other three immune indices did not differ between breeding phases. Red‐capped larks had higher nitric oxide concentrations than Rufous‐naped larks, which in turn had higher haptoglobin levels than red‐capped larks. T_max was higher during breeding than during non‐breeding for red‐capped larks only, suggesting potential confounding effect of T_max on the comparison of immune function between breeding and non‐breeding birds for this species. Overall, we conclude that in the two year‐round breeding equatorial larks, immune function is not down‐regulated during breeding.     

Idiopathic atrial fibrillation patients rapidly outgrow their low thromboembolic risk: a 10-year follow-up study

Background

Healthy atrial fibrillation (AF) patients will eventually outgrow their low thromboembolic risk. The purpose of this study is to compare the development of cardiovascular disease in healthy AF patients as compared to healthy sinus rhythm patients and to assess appropriate anticoagulation treatment.

Methods

Forty-one idiopathic paroxysmal AF patients (56 ± 10 years, 66% male) were compared with 45 healthy sinus rhythm patients. Patients were free of hypertension, antihypertensive and antiarrhythmic drugs, diabetes, congestive heart failure, coronary artery or peripheral vascular disease, previous stroke, thyroid, pulmonary and renal disease, and structural abnormalities on echocardiography.

Results

Baseline characteristics and echocardiographic parameters were the same in both groups. During 10.7 ± 1.6 years, cardiovascular disease and all-cause death developed significantly more often in AF patients as compared to controls (63% vs 31%, log rank p < 0.001). Even after the initial 5 years of follow-up, survival curves show divergent patterns (log rank p = 0.006). Mean duration to reach a CHA₂DS₂-VASc score > 1 among AF patients was 5.1 ± 3.0 years. Five of 24 (21%) patients with CHA₂DS₂-VASc > 1 did not receive oral anticoagulation therapy at follow-up. Mean duration of over- or undertreatment with oral anticoagulation in patients with CHA₂DS₂-VASc > 1 was 5 ± 3.0 years.

Conclusion

The majority of recently diagnosed healthy AF patients develop cardiovascular diseases with a consequent change in thromboembolic risk profile within a short time frame. A comprehensive follow-up of this patient category is necessary to avoid over- and undertreatment with anticoagulants.

     

Insights into lipid-protein interactions from computer simulations

Lipid-protein interactions play an important direct role in the function of many membrane proteins. We argue they are key players in membrane structure, modulate membrane proteins in more subtle ways than direct binding, and are important for understanding the mechanism of classes of hydrophobic drugs. By directly comparing membrane proteins from different families in the same, complex lipid mixture, we found a unique lipid environment for every protein. Extending this work, we identified both differences and similarities in the lipid environment of GPCRs, dependent on which family they belong to and in some cases their conformational state, with particular emphasis on the distribution of cholesterol. More recently, we have been studying modes of coupling between protein conformation and local membrane properties using model proteins. In more applied approaches, we have used similar methods to investigate specific hypotheses on interactions of lipid and lipid-like molecules with ion channels. We conclude this perspective with some considerations for future work, including a new more sophisticated coarse-grained force field (Martini 3), an interactive visual exploration framework, and opportunities to improve sampling.