Charge Compensation Mechanism of a Na+-coupled, Secondary Active Glutamate Transporter

Forward glutamate transport by the excitatory amino acid carrier EAAC1 is coupled to the inward movement of three Na(+) and one proton and the subsequent outward movement of one K(+) in a separate step. Based on indirect evidence, it was speculated that the cation binding sites bear a negative charge. However, little is known about the electrostatics of the transport process. Valences calculated using the Poisson-Boltzmann equation indicate that negative charge is transferred across the membrane when only one cation is bound. Consistently, transient currents were observed in response to voltage jumps when K(+) was the only cation on both sides of the membrane. Furthermore, rapid extracellular K(+) application to EAAC1 under single turnover conditions (K(+) inside) resulted in outward transient current. We propose a charge compensation mechanism, in which the C-terminal transport domain bears an overall negative charge of -1.23. Charge compensation, together with distribution of charge movement over many steps in the transport cycle, as well as defocusing of the membrane electric field, may be combined strategies used by Na(+)-coupled transporters to avoid prohibitive activation barriers for charge translocation.     

Consensus statement understanding health and malnutrition through a systems approach: the ENOUGH program for early life

Nutrition research, like most biomedical disciplines, adopted and often uses experimental approaches based on Beadle and Tatum’s one gene—one polypeptide hypothesis, thereby reducing biological processes to single reactions or pathways. Systems thinking is needed to understand the complexity of health and disease processes requiring measurements of physiological processes, as well as environmental and social factors, which may alter the expression of genetic information. Analysis of physiological processes with omics technologies to assess systems’ responses has only become available over the past decade and remains costly. Studies of environmental and social conditions known to alter health are often not connected to biomedical research. While these facts are widely accepted, developing and conducting comprehensive research programs for health are often beyond financial and human resources of single research groups. We propose a new research program on essential nutrients for optimal underpinning of growth and health (ENOUGH) that will use systems approaches with more comprehensive measurements and biostatistical analysis of the many biological and environmental factors that influence undernutrition. Creating a knowledge base for nutrition and health is a necessary first step toward developing solutions targeted to different populations in diverse social and physical environments for the two billion undernourished people in developed and developing economies.     

Label-free impedance detection of oligonucleotide hybridisation on interdigitated ultramicroelectrodes using electrochemical redox probes

The direct detection of oligodeoxynucleotide (ODN) hybridisation using electrochemical impedance spectroscopy was made on interdigitated array (IDA) gold (Au) ultramicroelectrodes manufactured by silicon technology. The immobilisation of single stranded ODNs (ssODNs) was accomplished by self-assembling of thiol-modified ODNs onto an Au-electrode surface. Faradaic impedance was measured in the presence of K(3)[Fe(CN)(6)]. Double strand formation was identified by a decrease of approximately 50% in impedance in the low frequency region in the presence of K(3)[Fe(CN)(6)], compared to the spectrum of single stranded ODN. The frequency dependent diffusion of Fe(CN)(6)(3-) ions through defects in the ODN monolayer determines the impedance of Au-ssODN surface. The influence of DNA intercalator methylene blue on the impedance of both, single and double strands, was examined along with K(3)[Fe(CN)(6)] and confirmed by cyclic voltammetry. The layer densities and the hybridisation have been further corroborated by chronoamperometric redox recycling of para-aminophenol (p-AP) in ELISA like experiments. It can be concluded, that a performed impedance spectroscopy did not change the layer density. The impedance spectroscopy at ultramicroelectrodes combined with faradaic redox reactions enhances the impedimetric detection of DNA hybridisation on IDA platforms.     

Flagellar motility and structure in the hyperthermoacidophilic archaeon Sulfolobus solfataricus

Flagellation in archaea is widespread and is involved in swimming motility. Here, we demonstrate that the structural flagellin gene from the crenarchaeaon Sulfolobus solfataricus is highly expressed in stationary-phase-grown cells and under unfavorable nutritional conditions. A mutant in a flagellar auxiliary gene, flaJ, was found to be nonmotile. Electron microscopic imaging of the flagellum indicates that the filaments are composed of right-handed helices.     

Effect of Nutrient Starvation on the Cellular Composition and Metabolic Capacity of Saccharomyces cerevisiae

This investigation addresses the following question: what are the important factors for maintenance of a high catabolic capacity under various starvation conditions? Saccharomyces cerevisiae was cultured in aerobic batch cultures, and during the diauxic shift cells were transferred and subjected to 24 h of starvation. The following conditions were used: carbon starvation, nitrogen starvation in the presence of glucose or ethanol, and both carbon starvation and nitrogen starvation. During the starvation period changes in biomass composition (including protein, carbohydrate, lipid, and nucleic acid contents), metabolic activity, sugar transport kinetics, and the levels of selected enzymes were recorded. Subsequent to the starvation period the remaining catabolic capacity was measured by addition of 50 mM glucose. The results showed that the glucose transport capacity is a key factor for maintenance of high metabolic capacity in many, but not all, cases. The results for cells starved of carbon, carbon and nitrogen, or nitrogen in the presence of glucose all indicated that the metabolic capacity was indeed controlled by the glucose transport ability, perhaps with some influence of hexokinase, phosphofructokinase, aldolase, and enolase levels. However, it was also demonstrated that there was no such correlation when nitrogen starvation occurred in the presence of ethanol instead of glucose.     

Immunochemical studies on the large polypeptide of electrophorus electroplax (Na+ + K+)-ATPase.

Antibodies against Lubrol-solubilized Electrophorus electroplax (Na+ + K+)-ATPase (ATP phosphohydrolase, EC 3.6.1.3) and its 96 000-dalton polypeptide (P96) were raised in rabbits. The P96 antibody does not cross react with the (Na+ + K+)-ATPase from mammalian species and tissues, but it cross reacts with the (Na+ + K+)-ATPase from both Electrophorus electroplax and brain. The combination of enzyme with anti-P96 is found to inhibit phosphoryl enzyme formation to the same extent that it inhibits enzyme activity. The rate of K+-sensitive dephosphorylation of phosphoryl enzyme appears to be unchanged. These are also found to be true with the antibody against the whole enzyme. Upon tryptic digestion of the enzyme-anti-P96 complex only the large polypeptide of the enzyme is protected. In the case of enzyme-anti-Lubrol-solubilized enzyme complex, both the large and small polypeptides are protected, whereas preimmune sera are without any protecting effect. The data indicate that the phosphoryl acceptor polypeptide and the Lubrol-solubilized electroplax (Na+ + K+)-ATPase from which the polypeptide is derived are phylogenetically distinct from those of the mammalian (Na+ + K+)-ATPases. The selective tryptic resistance of the enzyme-anti-P96 complex indicates that the two polypeptides are spatially well separated, possibly on opposite sides of the membrane.     

Distribution of Na+, K+-ATPase α-Subunit Isoforms in Rat Pituitary

The distributions of alpha-subunit isoforms of the Na+,K(+)-ATPase in rat pituitary were determined by immunoblotting and immunohistochemistry. Immunoreactivity for all three forms is present in the neural lobe, whereas the anterior lobe contains only alpha 1 and alpha 2. Most areas of the intermediate lobe exhibit faint immunoreactivity for only alpha 1, but thin strands of cells which stain strongly for all three isoforms are also present in this lobe. The previously reported ouabain inhibitable Na+,K(+)-ATPase activity in the neural lobe is consistent with the presence of both alpha 2 and alpha 3 subunits.     

Distribution of lecithin-cholesterol acyltransferase (LCAT) in human plasma lipoprotein fractions. Evidence for the association of active LCAT with low density lipoproteins

The distribution of lecithin-cholesterol acyltransferase (LCAT) in human plasma was assessed by measuring both LCAT mass and activity in plasma fractions separated by sequential flotation ultracentrifugation, single-spin gradient ultracentrifugation, dextran sulfate-Mg²⁺ precipitation or agarose gel filtration. Although most of the LCAT was found to be associated with the high density lipoprotein fraction, a small amount of active LCAT (approximately 1% of the plasma LCAT mass and activity) was consistently associated with the low density lipoprotein fraction. LCAT was not found in the very low density lipoprotein fraction. The LDL-associated LCAT may play an important role in the acylation of lysolecithin by lysolecithin acyltransferase activity of LCAT.     

Biphenyls as potent vitronectin receptor antagonists. Part 3: Squaric acid amides

Vitronectin receptor (alpha(V)beta(3)) antagonists have been implicated as a possible new treatment of restenosis following balloon angioplasty. In this work we investigate a series of novel arginine mimetic scaffolds leading to new insight of the alpha(V)beta(3)/ligand interaction. Squaric acid amide 10 is a subnanomolar alpha(V)beta(3) antagonist with improved potency on human smooth muscle cell migration.