Evolutionary analysis of the lysine-rich N-terminal cytoplasmic domains of the gastric H+,K+-ATPase and the Na+,K+-ATPase

The catalytic α-subunits of both the Na+,K+-ATPase and the gastric H+,K+-ATPase possess lysine-rich N-termini which project into the cytoplasm. Due to conflicting experimental results it is currently unclear whether the N-termini play a role in ion pump function or regulation, and, if they do, by what mechanism. Comparison of the lysine frequencies of the N-termini of both proteins with those of all of their extramembrane domains showed that the N-terminal lysine frequencies are far higher than one would expect simply from exposure to the aqueous solvent. The lysine frequency was found to vary significantly between different vertebrate classes, but this is due predominantly to a change in N-terminal length. As evidenced by a comparison between fish and mammals, an evolutionary trend towards an increase of the length of the N-terminus of the H+,K+-ATPase on going from an ancestral fish to mammals could be identified. This evolutionary trend supports the hypothesis that the N-terminus is important in ion pump function or regulation. In placental mammals, one of the lysines is replaced by serine (Ser-27), which is a target for protein kinase C. In most other animal species a lysine occupies this position and hence no protein kinase C target is present. Interaction with protein kinase C is thus not the primary role of the lysine-rich N-terminus. The disordered structure of the N-terminus may, via increased flexibility, facilitate interaction with another binding partner, e.g. the surrounding membrane, or help to stabilize particular enzyme conformations via the increased entropy it produces.Australian Research Counci

General and specific interactions of the phospholipid bilayer with P-type ATPases

Protein structure and function are modulated via interactions with their environment, representing both the surrounding aqueous media and lipid membranes that have an active role in shaping the structural topology of membrane proteins. Compared to a decade ago, there is now an abundance of crystal structural data on membrane proteins, which together with their functional studies have enhanced our understanding of the salient features of lipid-protein interactions. It is now important to recognize that membrane proteins are regulated by both: (1) general lipid-protein interactions, where the general physicochemical properties of the lipid environment affect the conformational flexibility of a membrane protein; and (2) by specific lipid-protein interactions, where lipid molecules directly interact via chemical interactions with specific lipid-binding sites located on the protein. However, due to local differences in membrane composition, thickness and lipid packing, local membrane physical properties and hence the associated lipid-protein interactions also differ due to membrane location, even for the same protein. Such a phenomenon has been shown to be true for one family of integral membrane ion pumps, the P2-type adenosine triphosphatases (ATPases). Despite being highly homologous, individual members of this family have distinct structural and functional activity and are an excellent candidate to highlight how the local membrane physical properties and specific lipid-protein interactions play a vital role in facilitating the structural rearrangements of these proteins necessary for their activity. Hence in this review, we focus on both the general and specific lipid-protein interactions and will mostly discuss the structure-function relationships of the following P2-type ATPases, Na+,K+-ATPase (NKA), gastric H+,K+-ATPase (HKA) and sarco(endo)plasmic reticulum Ca2+-ATPase (SERCA), in concurrence with their lipid environment.Australian Research Counci

Quantitative calculation of the role of the Na+,K+-ATPase in thermogenesis

AbstractThe Na+,K+-ATPase is accepted as an important source of heat generation (thermogenesis) in animals. Based on information gained on the kinetics of the enzyme's partial reactions we consider via computer simulation whether modifications to the function of the combined Na+,K+-ATPase/plasma membrane complex system could lead to an increased body temperature, either through the course of evolution or during an individual's lifespan. The enzyme's kinetics must be considered because it is the rate of heat generation which determines body temperature, not simply the amount of heat per enzymatic cycle. The results obtained indicate that a decrease in thermodynamic efficiency of the Na+,K+-ATPase, which could come about by Na+ substituting for K+ on the enzyme's extracellular face, could not account for increased thermogenesis. The only feasible mechanisms are an increase in the enzyme's expression level or an increase in its ion pumping activity. The major source of Na+,K+-ATPase-related thermogenesis (72% of heat production) is found to derive from passive Na+ diffusion into the cell, which counterbalances outward Na+ pumping to maintain a constant Na+ concentration gradient across the membrane. A simultaneous increase in both Na+,K+-ATPase activity and the membrane's passive Na+ permeability could promote a higher body temperature

Antibacterial Activity and Iron Release of Organic-Inorganic Hybrid Biomaterials Synthesized via the Sol-Gel Route

The aim of this work was the synthesis of hybrid materials of iron (II)-based therapeutic systems via the sol-gel method. Increasing amounts of polyethylene glycol (PEG 6, 12, 24, 50 wt%) were added to SiO2/Fe20 wt% to modulate the release kinetics of the drug from the systems. Fourier-transform infrared (FTIR) spectroscopy was used to study the interactions between different components in the hybrid materials. The release kinetics in a simulated body fluid (SBF) were investigated, and the amount of Fe2+ released was detected via ultraviolet-visible spectroscopy (UV-Vis) after reaction with ortho-phenanthroline. Furthermore, biological characterization was carried out. The bioactivity of the synthesized hybrid materials was evaluated via the formation of a layer of hydroxyapatite on the surface of samples soaked in SBF using spectroscopy. Finally, the potential antibacterial properties of seven different materials against two different bacteria—E. coli and S. aureus—were investigated

Initial Characteristics of Kepler Short Cadence Data

The Kepler Mission offers two options for observations -- either Long Cadence (LC) used for the bulk of core mission science, or Short Cadence (SC) which is used for applications such as asteroseismology of solar-like stars and transit timing measurements of exoplanets where the 1-minute sampling is critical. We discuss the characteristics of SC data obtained in the 33.5-day long Quarter 1 (Q1) observations with Kepler which completed on 15 June 2009. The truly excellent time series precisions are nearly Poisson limited at 11th magnitude providing per-point measurement errors of 200 parts-per-million per minute. For extremely saturated stars near 7th magnitude precisions of 40 ppm are reached, while for background limited measurements at 17th magnitude precisions of 7 mmag are maintained. We note the presence of two additive artifacts, one that generates regularly spaced peaks in frequency, and one that involves additive offsets in the time domain inversely proportional to stellar brightness. The difference between LC and SC sampling is illustrated for transit observations of TrES-2.Comment: 5 pages, 4 figures, ApJ Letters in pres

Mechanisms of cell uptake and toxicity of the anticancer drug cisplatin

Dieser Beitrag ist mit Zustimmung des Rechteinhabers aufgrund einer (DFG geförderten) Allianz- bzw. Nationallizenz frei zugänglich.This publication is with permission of the rights owner freely accessible due to an Alliance licence and a national licence (funded by the DFG, German Research Foundation) respectively.Two major issues which hamper the use of the anticancer drug cisplatin are the development of cancer cell resistance and its nephrotoxicity. One possible mechanism by which resistance is reported to develop is a reduction in drug uptake across the cell membrane. While the passive uptake of cisplatin has long been cited as an important contribution, far greater attention has been given to active modes of uptake, particularly in recent research. Using unilamellar lipid vesicles together with the stopped-flow kinetic method we show here that the permeability coefficient of cisplatin increases significantly with the chloride concentration of the medium. This supports the hypothesis that cisplatin can enter cells via passive permeation through the lipid phase of the membrane, but becomes trapped within the cytoplasm because dissociation of chloride ligands yields a membrane-impermeant positively-charged aqua derivative. This is important evidence for a major role of passive membrane diffusion in the uptake of cisplatin, and suggests that reduced cell uptake is unlikely to be a significant mechanism leading to the development of drug resistance. Studies of rubidium ion uptake into the cytoplasm of Xenopus oocytes via the Na+,K+-ATPase show significant inhibition of this ion pump when cisplatin is present in the cytoplasm. Because Na+,K+-ATPase activity is essential to the survival of all animal cells, e.g. via maintenance of cell volume, and the Na+,K+-ATPase is expressed at particularly high levels within the membranes of kidney tubules where it plays a crucial role in nutrient reabsorption, these results suggest that cisplatin-induced inhibition of the Na+,K+-ATPase is a likely contributing cause for the nephrotoxicity of cisplatin.DFG, EXC 314, Unifying Concepts in Catalysi

P3-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP for the Rapid Activation of the Na+,K+-ATPase

This is the published version, also available here: http://dx.doi.org/10.1016/S0006-3495(00)76387-9.P3-[2-(4-hydroxyphenyl)-2-oxo]ethyl ATP (pHP-caged ATP) has been investigated for its application as a phototrigger for the rapid activation of electrogenic ion pumps. The yield of ATP after irradiation with a XeCl excimer laser (λ = 308 nm) was determined at pH 6.0–7.5. For comparison, the photolytic yields of P3-[1-(2-nitrophenyl)]ethyl ATP (NPE-caged ATP) and P3-[1,2-diphenyl-2-oxo]ethyl ATP (desyl-caged ATP) were also measured. It was shown that at λ = 308 nm pHP-caged ATP is superior to the other caged ATP derivatives investigated in terms of yield of ATP after irradiation. Using time-resolved single-wavelength IR spectroscopy, we determined a lower limit of 106 s−1 for the rate constant of release of ATP from pHP-caged ATP at pH 7.0. Like NPE-caged ATP, pHP-caged ATP and desyl-caged ATP bind to the Na+,K+-ATPase and act as competitive inhibitors of ATPase function. Using pHP-caged ATP, we investigated the charge translocation kinetics of the Na+,K+-ATPase at pH 6.2–7.4. The kinetic parameters obtained from the electrical measurements are compared to those obtained with a technique that does not require caged ATP, namely parallel stopped-flow experiments using the voltage-sensitive dye RH421. It is shown that the two techniques yield identical results, provided the inhibitory properties of the caged compound are taken into account. Our results demonstrate that under physiological (pH 7.0) and slightly basic (pH 7.5) or acidic (pH 6.0) conditions, pHP-caged ATP is a rapid, effective, and biocompatible phototrigger for ATP-driven biological systems

Shape analysis of the StW 578 calotte from Jacovec Cavern, Gauteng (South Africa)

The fossiliferous deposits within the lower-lying Jacovec Cavern in the locality of Sterkfontein yielded valuable hominin remains, including the StW 578 specimen. Because StW 578 mainly preserves the calotte, the taxonomic status of this specimen has been a matter of discussion. Within this context, here we employed high-resolution microtomography and a landmark-free registration method to explore taxonomically diagnostic features in the external surface of the StW 578 calotte. Our comparative sample included adult humans and common chimpanzees as well as one Australopithecus africanus specimen (Sts 5). We partially restored the StW 578 calotte digitally and compared it to extant specimens and Sts 5 using a landmark-free registration based on smooth and invertible surface deformation. Our comparative shape analysis reveals morphological differences with extant humans, especially in the frontal bones, and with extant chimpanzees, as well as intriguing specificities in the morphology of the StW 578 parietal bones. Lastly, our study suggests morphological proximity between StW 578 and Sts 5. Given the intimate relationship between the brain and the braincase, as well as the integration of the hominin face and neurocranium, we suggest that cranial vault shape differences between StW 578 and extant humans, if confirmed by further analyses, could be either explained by differences in brain surface morphology or in the face. Besides providing additional information about the morphology of the Jacovec calotte that will be useful in future taxonomic discussion, this study introduces a new protocol for the landmark-free analysis of fossil hominin cranial shape.Significance:• We provide further information on the enigmatic fossil specimen StW 578.• We introduce a new approach for the morphological study of fossil hominin crania.• We highlight morphological similarities between StW 578 and ‘Mrs Ples’

Characterization of early disease status in treatment-naive male paediatric patients with Fabry disease enrolled in a randomized clinical trial.

Trial designThis analysis characterizes the degree of early organ involvement in a cohort of oligo-symptomatic untreated young patients with Fabry disease enrolled in an ongoing randomized, open-label, parallel-group, phase 3B clinical trial.MethodsMales aged 5-18 years with complete α-galactosidase A deficiency, without symptoms of major organ damage, were enrolled in a phase 3B trial evaluating two doses of agalsidase beta. Baseline disease characteristics of 31 eligible patients (median age 12 years) were studied, including cellular globotriaosylceramide (GL-3) accumulation in skin (n = 31) and kidney biopsy (n = 6; median age 15 years; range 13-17 years), renal function, and glycolipid levels (plasma, urine).ResultsPlasma and urinary GL-3 levels were abnormal in 25 of 30 and 31 of 31 patients, respectively. Plasma lyso-GL-3 was elevated in all patients. GL-3 accumulation was documented in superficial skin capillary endothelial cells (23/31 patients) and deep vessel endothelial cells (23/29 patients). The mean glomerular filtration rate (GFR), measured by plasma disappearance of iohexol, was 118.1 mL/min/1.73 m(2) (range 90.4-161.0 mL/min/1.73 m(2)) and the median urinary albumin/creatinine ratio was 10 mg/g (range 4.0-27.0 mg/g). On electron microscopy, renal biopsy revealed GL-3 accumulation in all glomerular cell types (podocytes and parietal, endothelial, and mesangial cells), as well as in peritubular capillary and non-capillary endothelial, interstitial, vascular smooth muscle, and distal tubules/collecting duct cells. Lesions indicative of early Fabry arteriopathy and segmental effacement of podocyte foot processes were found in all 6 patients.ConclusionsThese data reveal that in this small cohort of children with Fabry disease, histological evidence of GL-3 accumulation, and cellular and vascular injury are present in renal tissues at very early stages of the disease, and are noted before onset of microalbuminuria and development of clinically significant renal events (e.g. reduced GFR). These data give additional support to the consideration of early initiation of enzyme replacement therapy, potentially improving long-term outcome.Trial registrationClinicalTrials.gov NCT00701415

Mechanism of Action of Surface Immobilized Antimicrobial Peptides Against Pseudomonas aeruginosa

Bacterial colonization and biofilm development on medical devices can lead to infection. Antimicrobial peptide-coated surfaces may prevent such infections. Melimine and Mel4 are chimeric cationic peptides showing broad-spectrum antimicrobial activity once attached to biomaterials and are highly biocompatible in animal models and have been tested in Phase I and II/III human clinical trials. These peptides were covalently attached to glass using an azidobenzoic acid linker. Peptide attachment was confirmed using X-ray photoelectron spectroscopy and amino acid analysis. Mel4 when bound to glass was able to adopt a more ordered structure in the presence of bacterial membrane mimetic lipids. The ability of surface bound peptides to neutralize endotoxin was measured along with their interactions with the bacterial cytoplasmic membrane which were analyzed using DiSC(3)-5 and Sytox green, Syto-9, and PI dyes with fluorescence microscopy. Leakage of ATP and nucleic acids from cells were determined by analyzing the surrounding fluid. Attachment of the peptides resulted in increases in the percentage of nitrogen by 3.0% and 2.4%, and amino acid concentrations to 0.237 nmole and 0.298 nmole per coverslip on melimine and Mel4 coated surfaces, respectively. The immobilized peptides bound lipopolysaccharide and disrupted the cytoplasmic membrane potential of Pseudomonas aeruginosa within 15 min. Membrane depolarization was associated with a reduction in bacterial viability by 82% and 63% for coatings melimine and Mel4, respectively (p < 0.001). Disruption of membrane potential was followed by leakage of ATP from melimine (1.5 ± 0.4 nM) or Mel4 (1.3 ± 0.2 nM) coated surfaces compared to uncoated glass after 2 h (p < 0.001). Sytox green influx started after 3 h incubation with either peptide. Melimine coatings yielded 59% and Mel4 gave 36% PI stained cells after 4 h. Release of the larger molecules (DNA/RNA) commenced after 4 h for melimine (1.8 ± 0.9 times more than control; p = 0.008) and after 6 h with Mel4 (2.1 ± 0.2 times more than control; p < 0.001). The mechanism of action of surface bound melimine and Mel4 was similar to that of the peptides in solution, however, their immobilization resulted in much slower (approximately 30 times) kinetics