Theoretical model of interactions between ligand-binding sites in a dimeric protein and its application for the analysis of thiamine diphosphate binding to yeast transketolase

The binding of thiamin diphosphate (ThDP) to yeast dimeric apotransketolase (apoTK) is accompanied by the appearance of a band in the absorption spectrum with maximum at 320 nm. The saturation function has been analyzed using a scheme that involves binding of ThDP to each subunit followed by the conformational transition of this subunit. It is assumed that the binding of ThDP to one subunit may affect the conformational transition of the other subunit. Rigorous mathematical expressions describing the dependence of the optical absorption on the total concentration of ThDP are first developed. Equilibrium constants and corresponding rate constants for the binding of ThDP to apoTK have been estimated. The negative cooperativity in the ThDP binding has been characterized by the function reflecting the dependence of the conformational change on the saturation of apoTK by ThDP.     

Spatial patterns formed by chemotactic bacteria Escherichia coli

In certain experimental conditions, bacteria form complex spatial-temporal patterns. A striking example of such kind was reported by Budrene and Berg (1991), who observed a wide variety of different colony structures ranging from arrays of spots to radially oriented stripes or arrangements of more complex elongated spots, formed by Escherichia coli. We discuss the relevant mechanisms of intercellular regulation in bacterial colony which may cause pattern formation, and formulate the corresponding mathematical model. In numerical experiments a variety of patterns, observed in real systems, is reproduced. The dynamics of their formation is investigated.     

Bafilomycin A1 activates HIF-dependent signalling in human colon cancer cells via mitochondrial uncoupling

Mitochondrial uncoupling is implicated in many patho(physiological) states. Using confocal live cell imaging and an optical O₂ sensing technique, we show that moderate uncoupling of the mitochondria with plecomacrolide Baf (bafilomycin A1) causes partial depolarization of the mitochondria and deep sustained deoxygenation of human colon cancer HCT116 cells subjected to 6% atmospheric O₂. A decrease in iO₂ (intracellular O₂) to 0–10 μM, induced by Baf, is sufficient for stabilization of HIFs (hypoxia inducible factors) HIF-1α and HIF-2α, coupled with an increased expression of target genes including GLUT1 (glucose transporter 1), HIF PHD2 (prolyl hydroxylase domain 2) and CAIX (carbonic anhydrase IX). Under the same hypoxic conditions, treatment with Baf causes neither decrease in iO₂ nor HIF-α stabilization in the low-respiring HCT116 cells deficient in COX (cytochrome c-oxidase). Both cell types display equal capacities for HIF-α stabilization by hypoxia mimetics DMOG (dimethyloxalylglycine) and CoCl₂, thus suggesting that the effect of Baf under hypoxia is driven mainly by mitochondrial respiration. Altogether, by activating HIF signalling under moderate hypoxia, mitochondrial uncoupling can play an important regulatory role in colon cancer metabolism and modulate adaptation of cancer cells to natural hypoxic environments.     

Self-assembly and biphasic iron-binding characteristics of Mms6, a bacterial protein that promotes the formation of superparamagnetic magnetite nanoparticles of uniform size and shape

Highly ordered mineralized structures created by living organisms are often hierarchical in structure with fundamental structural elements at nanometer scales. Proteins have been found responsible for forming many of these structures, but the mechanisms by which these biomineralization proteins function are generally poorly understood. To better understand its role in biomineralization, the magnetotactic bacterial protein, Mms6, which promotes the formation in vitro of superparamagnetic magnetite nanoparticles of uniform size and shape, was studied for its structure and function. Mms6 is shown to have two phases of iron binding: one high affinity and stoichiometric and the other low affinity, high capacity, and cooperative with respect to iron. The protein is amphipathic with a hydrophobic N-terminal domain and hydrophilic C-terminal domain. It self-assembles to form a micelle, with most particles consisting of 20-40 monomers, with the hydrophilic C-termini exposed on the outside. Studies of proteins with mutated C-terminal domains show that the C-terminal domain contributes to the stability of this multisubunit particle and binds iron by a mechanism that is sensitive to the arrangement of carboxyl/hydroxyl groups in this domain.     

Genetically determined P2X7 receptor pore formation regulates variability in chronic pain sensitivity

Chronic pain is highly variable between individuals, as is the response to analgesics. Although much of the variability in chronic pain and analgesic response is heritable, an understanding of the genetic determinants underlying this variability is rudimentary. Here we show that variation within the coding sequence of the gene encoding the P2X7 receptor (P2X7R) affects chronic pain sensitivity in both mice and humans. P2X7Rs, which are members of the family of ionotropic ATP-gated receptors, have two distinct modes of function: they can function through their intrinsic cationic channel or by forming nonselective pores that are permeable to molecules with a mass of up to 900 Da. Using genome-wide linkage analyses, we discovered an association between nerve-injury-induced pain behavior (mechanical allodynia) and the P451L mutation of the mouse P2rx7 gene, such that mice in which P2X7Rs have impaired pore formation as a result of this mutation showed less allodynia than mice with the pore-forming P2rx7 allele. Administration of a peptide corresponding to the P2X7R C-terminal domain, which blocked pore formation but not cation channel activity, selectively reduced nerve injury and inflammatory allodynia only in mice with the pore-forming P2rx7 allele. Moreover, in two independent human chronic pain cohorts, a cohort with pain after mastectomy and a cohort with osteoarthritis, we observed a genetic association between lower pain intensity and the hypofunctional His270 (rs7958311) allele of P2RX7. Our findings suggest that selectively targeting P2X7R pore formation may be a new strategy for individualizing the treatment of chronic pain.     

Functional neuronal cells generated by human parthenogenetic stem cells

Parent of origin imprints on the genome have been implicated in the regulation of neural cell type differentiation. The ability of human parthenogenetic (PG) embryonic stem cells (hpESCs) to undergo neural lineage and cell type-specific differentiation is undefined. We determined the potential of hpESCs to differentiate into various neural subtypes. Concurrently, we examined DNA methylation and expression status of imprinted genes. Under culture conditions promoting neural differentiation, hpESC-derived neural stem cells (hpNSCs) gave rise to glia and neuron-like cells that expressed subtype-specific markers and generated action potentials. Analysis of imprinting in hpESCs and in hpNSCs revealed that maternal-specific gene expression patterns and imprinting marks were generally maintained in PG cells upon differentiation. Our results demonstrate that despite the lack of a paternal genome, hpESCs generate proliferating NSCs that are capable of differentiation into physiologically functional neuron-like cells and maintain allele-specific expression of imprinted genes. Thus, hpESCs can serve as a model to study the role of maternal and paternal genomes in neural development and to better understand imprinting-associated brain diseases.     

Delivery of the Cu-transporting ATPase ATP7B to the plasma membrane in Xenopus oocytes

Cu-transporting ATPase ATP7B (Wilson disease protein) is essential for the maintenance of intracellular copper concentration. In hepatocytes, ATP7B is required for copper excretion, which is thought to occur via a transient delivery of the ATP7B- and copper-containing vesicles to the apical membrane. The currently available experimental systems do not allow analysis of ATP7B at the cell surface. Using epitope insertion, we identified an extracellular loop into which the HA-epitope can be introduced without inhibiting ATP7B activity. The HA-tagged ATP7B was expressed in Xenopus oocytes and the presence of ATP7B at the plasma membrane was demonstrated by electron microscopy, freeze-fracture experiments, and surface luminescence measurements in intact cells. Neither the deletion of the entire N-terminal copper-binding domain nor the inactivating mutation of catalytic Asp1027 affected delivery to the plasma membrane of oocytes. In contrast, surface targeting was decreased for the ATP7B variants with mutations in the ATP-binding site or the intra-membrane copper-binding site, suggesting that ligand-stabilized conformation(s) are important for ATP7B trafficking. The developed system provides significant advantages for studies that require access to both sides of ATP7B in the membrane.     

Effect of snow depth on under-ice irradiance and growth of <Emphasis Type="Italic">Aulacoseira baicalensis</Emphasis> in Lake Baikal

Lake Baikal freezes for 4–5 months each year; yet the planktonic diatoms that grow under the ice include some of the largest species found in freshwater. An important factor influencing their growth is the depth of snow. In this study, a population of Aulacoseira baicalensis developed where there was little or no snow on the ice but declined where there was 10 cm of snow, because 99% of the available light was attenuated. Culture studies of light response showed that A. baicalensis was adapted to relatively low light intensities (<40 μmol m⁻² s⁻¹) that were close to the average that a cell experiences in L. Baikal when mixed vertically by convection to depths that can exceed 100 m. On sunny days, cell division could be inhibited down to >10 m depth but narrow (<15 μm) diameter cells trapped in high light intensities in sub-ice layers switched to auxosporulation and size regeneration.