Crystal structure of the sodium-potassium pump (Na+,K+-ATPase) with bound potassium and ouabain

The sodium-potassium pump (Na⁺,K⁺-ATPase) is responsible for establishing Na⁺ and K⁺ concentration gradients across the plasma membrane and therefore plays an essential role in, for instance, generating action potentials. Cardiac glycosides, prescribed for congestive heart failure for more than 2 centuries, are efficient inhibitors of this ATPase. Here we describe a crystal structure of Na⁺,K⁺-ATPase with bound ouabain, a representative cardiac glycoside, at 2.8 Å resolution in a state analogous to E2·2K⁺·Pi. Ouabain is deeply inserted into the transmembrane domain with the lactone ring very close to the bound K⁺, in marked contrast to previous models. Due to antagonism between ouabain and K⁺, the structure represents a low-affinity ouabain-bound state. Yet, most of the mutagenesis data obtained with the high-affinity state are readily explained by the present crystal structure, indicating that the binding site for ouabain is essentially the same. According to a homology model for the high affinity state, it is a closure of the binding cavity that confers a high affinity.     

Dose–response relationship in inferior oblique muscle recession

BACKGROUND: Recession of the inferior oblique muscle is a widely applied operation in the treatment of strabismus sursoadductorius. In this retrospective study, the dose-response relationship of this procedure was determined in order to improve surgical outcomes. In particular, the effect of an additional anteroposition of the operated muscle was analysed, as well as differences between short and long term results. METHODS: This retrospective study included 37 patients. Inclusion criteria were unilateral strabismus sursoadductorius and normal retinal correspondence. Exclusion criteria comprised previous ocular muscle surgery and any ocular or orbital disease. Nineteen patients had received an additional anteroposition of the muscle. Patients were examined at a tangent screen pre-operatively and 1 day post-operatively (short-term effect). Sixteen patients underwent an additional examination 3 months post-operatively (long-term effect). Changes of vertical, torsional and horizontal deviations, measured in different positions of gaze, were related to the surgical dose in order to calculate the dose-response relationship by linear regression analysis. RESULTS: Surgery always reduced preoperative deviations. The short-term vertical dose-response without/with anteroposition was 0.6 degrees /0.5 degrees per mm in primary gaze and 1.2 degrees /1.1 degrees per mm in adduction. The short-term torsional dose-response without/with anteroposition was 0.8 degrees /0.5 degrees per mm in primary gaze. Three months later, the vertical effect had remained constant, whereas the torsional effect had decreased to 0.6 degrees /0.1 degrees per mm. Variance was high, e.g. the standard deviation of the short-term effect for the vertical deviation in adduction was 2.5 degrees . CONCLUSIONS: Despite the large variation of effects, a dose-response relationship could be established, facilitating surgical planning. Large vertical deviations with small excyclodeviation are an indication for additional anteropositioning. The torsional effect of inferior oblique muscle recessions can diminish over time.     

Using biorelevant dissolution to obtain IVIVC of solid dosage forms containing a poorly-soluble model compound

The usefulness of selected biorelevant dissolution media (BDM) to predict in vivo drug absorption was studied. Dissolution profiles of solid formulations of a poorly soluble model compound were compared in BDM simulating fasted and two levels of fed state. A non-physiologically relevant medium containing the cationic surfactant, cetrimide, was also investigated. All the media studied were capable of differentiating between the formulations employed, with formulation A consistently ranking high and formulations C and D ranking low. An in vivo dog study was carried out and an attempt was made to obtain a level A correlation between the plasma absorption curves and in vitro dissolution curves, using non-linear regression software. The in vitro-in vivo correlation (IVIVC) models developed indicated that fed state media (BDM 3) containing high levels of both bile salts (BS) and lipolysis products (LP) were best able to predict in vivo pharmacokinetic parameters (Cmax and AUC) with prediction errors lower than 10%. Overall, design and use of appropriate media for in vitro dissolution is extremely important. This study demonstrates the potential of physiologically relevant media containing both BS and LP for use in formulation and early drug development.     

In vivo assessment of mutations in the phenylalanine hydroxylase gene by phenylalanine loading: Characterization of seven common mutations

Abstract Mutations in the gene encoding phenylalanine hydroxylase (PAH) cause persistent hyperphenyl-alaninaemia. To date, more than 200 point mutations and microdeletions have been characterized. Each mutation has a particular quantitative effect on enzyme activity and recessive expression of different mutant alleles results in a marked interindividual heterogeneity of metabolic and clinical phenotypes. In this paper we demonstrate how a simple clinical test can be used to evaluate the correlation between mutation genotype and phenylalanine metabolism. In hyperphenylalaninaemic patients with known PAH mutation genotype, we have investigated phenylalanine turnover in vivo by measuring the ability to eliminate a test dose ofl-phenyl-alanine. All patients could be considered functionally hemizygous for one of their mutant alleles by carrying on the other allele a mutation that is known to completely abolish PAH activity and encode a peptide with no immunoreactivity. Seven mutations (R408W, IVS-12nt1, R261Q, G46S, Y414C, A104D, and D415N) were characterized by oral phenylalanine loading, each mutation being represented by at least three patients. The elimination profile determined for a 3-day period provides a measure to compare residual activity of the mutant proteins and to assign each mutation to a particular metabolic phenotype. The established relation between genotype and phenotype may enable prediction of the severity of the disease by genotype determination in the newborn period. This will aid in the management of hyperphenylalaninaemia and may improve prognosis.Conclusion The possibility of predicting the residual enzyme activity by DNA analysis performed already in the newborn period allows the prompt implementation of a diet that is adjusted to the degree of PAH deficiency. This may improve management and prognosis of hyperphenylalaninaemia.     

Optic disk drusen

Optic disk drusen occur in 3.4 to 24 per 1,000 population and are bilateral in approximately 75%. Disturbance in the axonal metabolism in the presence of a small scleral canal—regardless of eyelength—is considered responsible for the development. The drusen increase in size, becoming more visible with age due to continuing calcium apposition, and they are associated with visual field defects in a considerable number of patients. Patients do not usually notice these defects, despite their progressive nature over the years, and this indicates an insidious course. A correct diagnosis of optic disk drusen is mandatory, although effective treatment is not yet available. It is most important to differentiate optic disk drusen from papilledema in order to avoid unnecessary neurological examinations, but also to avoid overlooking genuine neurologic disorders. Because optic disk drusen can cause severe visual field defects, patients require individual consultation regarding work issues and whether or not to drive. Optic disk drusen can be accompanied by vascular complications as well. In some cases these vascular changes—for example, choroidal neovascularization—are treatable. Patients with optic disk drusen should undergo regular visual field, IOP, and nerve fiber layer examinations. In patients with deteriorating visual field and borderline IOP, we recommend antiglaucomatous therapy.     

Fungal flora in groundwater-derived public drinking water

In order to assess the dissemination of hygienically relevant fungi via the public drinking water distribution system, a 12-month survey was performed on groundwater-derived drinking water from 29 water supplies in North Rhine-Westphalia, Germany. Frequencies of contaminated water samples, and the prevalent species and patterns of occurrence in raw water, waterworks, the network and house installations were studied on the basis of 2657 water samples. Results were obtained by long-term incubation of 1 ml aliquots of water samples on agar-based culture media, following bacteriological procedures documented in the German drinking water regulations (Anon, 1990). No correlation with standard hygiene indicators, such as E. coli or other coliform bacteria was observed. Common opportunistic and allergenic Aspergillus species were encountered only rarely. The fungal flora was dominated by a limited number of species of Acremonium, Exophiala, Penicillium and particularly Phialophora; some of them occurred throughout the entire drinking water system and are thought to constitute a resident fungal flora. Phialophora sp. nov., to be described as a new species elsewhere, was ubiquitous; it was found in 26.6% of the samples positive for fungi (7.5% of 2657). Fungal diversity in the network itself was significantly lower than in raw water and house installations, indicating that not all fungi gaining access to the system are capable of surviving for longer periods. For species such as Verticillium lecanii, found exclusively after the introduction of newly buried pipes and remaining localized at those sites, introduction via arthropod vectors is likely. The resident species of Phialophora, Exophiala and Acremonium are particularly significant as they are shown to be disseminated efficiently by public drinking water.     

Strong association between house characteristics and malaria vectors in Sri Lanka

The objective of this study was to determine whether house characteristics could be used to further refine the residual insecticide-spraying program in Sri Lanka. Indoor-resting mosquito densities were estimated in 473 houses based on fortnightly collections over a two-and-a-half-year period. The type of house construction and the exact location of all houses were determined. In a multivariate analysis, distance of less than 750 meters between a house and the main vector-breeding site was strongly associated with the presence of Anopheles culicifacies in the house (odds ratio [OR] 4.8, 95% confidence interval [CI] 3.4-6.8) and to a lesser extent with the presence of An. subpictus (OR 1.4, 95% CI 1.1-1.7). Poor housing construction also was an independent risk factor (OR for An. culicifacies 1.3, 95% CI 1.0-1.9; OR for An. subpictus 1.3, 95% CI 1.0-1.6). It is recommended that a malaria control strategy focus on residential areas within 750 meters of streams and rivers, with special attention given to areas with the poorest type of house construction.