Functional analysis of polar amino-acid residues in membrane associated regions of the NHE1 isoform of the mammalian Na+/H+ exchanger.

The NHE1 isoform of the Na+/H+ exchanger is a ubiquitous plasma membrane protein that regulates intracellular pH in mammalian cells. Site-specific mutagenesis was used to examine the functional role of conserved, polar amino-acid residues occurring in segments of the protein associated with the membrane. Seventeen mutant proteins were assessed by characterization of intracellular pH changes in stably transfected cells that lacked an endogenous Na+/H+ exchanger. All of the mutant proteins were targeted correctly to the plasma membrane and were expressed at similar levels. Amino-acid residues Glu262 and Asp267 were critical to Na+/H+ exchanger activity while mutation of Glu391 resulted in only a partial reduction in activity. The Glu262-->Gln mutant was expressed partially as a deglycosylated protein with increased sensitivity to trypsin treatment in presence of Na+. Substitution of mutated Glu262, Asp267 and Glu391 with alternative acidic residues restored Na+/H+ exchanger activity. The Glu262-->Asp mutant had a decreased affinity for Li+, but its activity for Na+ and H+ ions was unaffected. The results support the hypothesis that side-chain oxygen atoms in a few, critically placed amino acids are important in Na+/H+ exchanger activity and the acidic amino-acid residues at positions 262, 267 and 391 are good candidates for being involved in Na+ coordination by the protein.     

Biodiversity and ecology of epilithic diatoms in the Agnéby River,Ivory Coast

The ecology and taxonomy of the epilithic diatom flora of the Agnéby River, Ivory Coast were studied in 2012. Ten sites were investigated and diatoms were sampled on glass slides immersed for a period of 30 days during the wet and dry seasons. Physico-chemical parameters were measured at each site while sampling diatoms. Five taxa were largely dominant: Planothidium comperei CE Wetzel, N’Guessan and Tison-Rosebery, Eolimna minima (Grunow) Lange-Bertalot, Planothidium piaficum (JR Carter and Denny) CE Wetzel and Ector, Cocconeis schroederi Foged and Cocconeis scutellum var. parva (Grunow in Van Heurck) Cleve. Electrical conductivity, temperature, dissolved oxygen, nitrite and phosphorus were found to influence the distribution of taxa.     

Membrane-protein interactions contribute to efficient 27-hydroxylation of cholesterol by mitochondrial cytochrome P450 27A1

Mitochondrial cytochrome P450 27A1 (P450 27A1) catalyzes 27-hydroxylation of cholesterol, the first step in the alternative bile acid biosynthetic pathway. Although several crystal structures of P450s are known, no structural information is available for the mammalian, membrane-bound enzymes involved in the removal of cholesterol from the body. We prepared a three-dimensional model of P450 27A1 based on the structure of P450 BM-3. Conservative and non-conservative mutations were introduced at hydrophobic and positively charged residues in the putative F-G loop and the adjacent helix G (positions 219-237). Subcellular distribution of the mutant P450s expressed in Escherichia coli was used as a measure of membrane-protein interactions. Conservative substitutions of residues located on the surface, according to our model, L219V, L219I, Y220F, F223Y, L224I, R229K, V231L, F234Y, K236R, and R237K, weakened the association of the mutant P450s with the membrane and led to the appearance of up to 21% of P450 27A1 in the bacterial cytosol. It is likely that the mutated side chains are involved in binding to membrane phospholipids. Substitutions in the F-G loop did not significantly affect the K(m) value for cholesterol hydroxylation. However, non-conservative mutants, L219N, Y220A, Y220S, F223A, K226R, and R229A, had significantly impaired catalytic properties, indicating strict requirements for the size and polarity of the side chains at these positions for the catalysis. The results provide insight into the membrane topology of mitochondrial P450s and indicate the importance of membrane-protein interactions in the efficiency of reactions catalyzed by P450 27A1.     

Aqueous and salt solutions of quinine of low concentrations: Self-organization,physicochemical properties and actions on the electrical characteristics of neurons

Self-organization, the physicochemical properties of aqueous and salt solutions of quinine and the effects of salt quinine solutions in a wide range of concentrations (1 · 10^?22?1 · 10^?3 M) on the electrical characteristics of the edible snail’s identified neurons were studied. Similar non-monotonic concentration dependencies of physicochemical properties of aqueous and salt quinine solutions at low concentrations are obtained. This allows of predicting the occurrence of biological effects at low concentrations of quinine solutions. Intrinsic (within 5% of the interval) changes in membrane potential, the amplitude and duration of the neuron action potential under the influence of quinine salt solutions at concentrations of quinine of 1 · 10^?20, 1 · 10^?18, 1 · 10^?10 M are found. For these concentrations the extreme values of specific conductivity and pH are shown.     

Effects of sterol-binding agent nystatin on wheat roots: the changes in membrane permeability, sterols and glycoceramides

Plant sterols are important multifunctional lipids, which are involved in determining membrane properties. Biophysical characteristics of model lipid and isolated animal membranes with altered sterol component have been intensively studied. In plants however, the precise mechanisms of involvement of sterols in membrane functioning remain unclear. In present work the possible interactions between sterols and other membrane lipids in plant cells were studied. A useful experimental approach for elucidating the roles of sterols in membrane activity is to use agents that specifically bind with endogenous sterols, for example the antibiotic nystatin. Membrane characteristics and the composition of membrane lipids in the roots of wheat (Triticum aestivum L.) seedlings treated with nystatin were analyzed. The application of nystatin greatly increased the permeability of the plasma membrane for ions and SH-containing molecules and decreased the total sterol level mainly as a consequence of a reduction in the amount of β-sitosterol and campesterol. Dynamic light-scattering was used to confirm the in vitro formation of stable complexes between nystatin and β-sitosterol or cholesterol. Sterol depletion was accompanied by a significant rise in total glycoceramide (GlCer) content after 2h treatment with nystatin. Analysis of the GlCer composition using mass spectrometry with electrospray ionization demonstrated that nystatin induced changes in the ratio of molecular species of GlCer. Our results suggest that changes in the sphingolipid composition can contribute to the changes in plasma membrane functioning induced by sterol depletion.     

Genome-wide methylation and expression differences in HPV(+) and HPV(?) squamous cell carcinoma cell lines are consistent with divergent mechanisms of carcinogenesis

Oncogenic human papillomaviruses (HPV) are associated with nearly all cervical cancers and are increasingly important in the etiology of oropharyngeal tumors. HPV-associated head and neck squamous cell carcinomas (HNSCC) have distinct risk profiles and appreciate a prognostic advantage compared to HPV-negative HNSCC. Promoter hypermethylation is widely recognized as a mechanism in the progression of HNSCC, but the extent to which this mechanism is consistent between HPV(+) and HPV(−) tumors is unknown. To investigate the epigenetic regulation of gene expression in HPV-induced and carcinogen-induced cancers, we examined genome-wide DNA methylation and gene expression in HPV(+) and HPV(−) SCC cell lines. We used two platforms: the Illumina Infinium Methylation BeadArray and tiling arrays, and confirmed illustrative examples with pyrosequencing and quantitative PCR. These analyses indicate that HPV(+) cell lines have higher DNA methylation in genic and LINE-1 regions than HPV(−) cell lines. Differentially methylated loci between HPV(+) and HPV(−) cell lines significantly correlated with HPV-typed HNSCC primary tumor DNA methylation levels. Novel findings include higher promoter methylation of polycomb repressive complex 2 target genes in HPV(+) cells compared to HPV(−) cells and increased expression of DNMT3A in HPV(+) cells. Additionally, CDKN2A and KRT8 were identified as interaction hubs among genes with higher methylation and lower expression in HPV(−) cells. Conversely, RUNX2, IRS-1 and CCNA1 were major hubs with higher methylation and lower expression in HPV(+) cells. Distinct HPV(+) and HPV(−) epigenetic profiles should provide clues to novel targets for development of individualized therapeutic strategies.Key words: epigenetics, human papillomavirus, HNSCC, DNA methylation, squamous cell carcinoma, gene expression, microarrays, illumina infinium humanmethylation27 beadarray