Expression of the activity of cystine/glutamate exchange transporter,system x(c)(-), by xCT and rBAT

The expression of the activity of cystine/glutamate exchange transporter, designated system x(c)(-), requires two components, xCT and 4F2 heavy chain (4F2hc) in Xenopus oocytes. rBAT (related to b(0,+) amino acid transporter) has a significant homology to 4F2hc and is known to be located in the apical membrane of epithelial cells. To determine whether xCT can associate with rBAT and express the activity of system x(c)(-), xCT, and rBAT were co-expressed in Xenopus oocytes and in mammalian cultured cells. In the oocytes injected with rBAT cRNA alone, the activities of cystine and arginine transport were induced, indicating that the system b(0,+)-like transporter was expressed by associating the exogenous rBAT with an endogenous b(0,+)AT-like factor as reported previously. In the oocytes injected with xCT and rBAT cRNAs, the activity of cystine transport was further induced. This induced activity of cystine transport was partially inhibited by glutamate or arginine and completely inhibited by adding both amino acids. In these oocytes, the activity of glutamate transport was also induced and it was strongly inhibited by cystine. In NIH3T3 cells transfected with xCT cDNA alone, the activity of cystine transport was significantly increased, and in the cells transfected with both xCT and rBAT cDNAs, the activity of cystine transport was further enhanced. The enhanced activity was Na(+)-independent and was inhibited by glutamate and homocysteate. These results indicate that rBAT can replace 4F2hc in the expression of the activity of system x(c)(-) and suggest that system x(c)(-) activity could be expressed in the apical membrane of epithelial cells.     

The acid-base transport proteins NHE1 and NBCn1 regulate cell cycle progression in human breast cancer cells

Precise acid-base homeostasis is essential for maintaining normal cell proliferation and growth. Conversely, dysregulated acid-base homeostasis, with increased acid extrusion and marked extracellular acidification, is an enabling feature of solid tumors, yet the mechanisms through which intra- and extracellular pH (pH_i, pH_e) impact proliferation and growth are incompletely understood. The aim of this study was to determine the impact of pH, and specifically of the Na⁺/H⁺ exchanger NHE1 and Na⁺, HCO₃^? transporter NBCn1, on cell cycle progression and its regulators in human breast cancer cells. Reduction of pH_e to 6.5, a common condition in tumors, significantly delayed cell cycle progression in MCF-7 human breast cancer cells. The NHE1 protein level peaked in S phase and that of NBCn1 in G2/M. Steady state pH_i changed through the cell cycle, from 7.1 in early S phase to 6.8 in G2, recovering again in M phase. This pattern, as well as net acid extrusion capacity, was dependent on NHE1 and NBCn1. Accordingly, knockdown of either NHE1 or NBCn1 reduced proliferation, prolonged cell cycle progression in a manner involving S phase prolongation and delayed G2/M transition, and altered the expression pattern and phosphorylation of cell cycle regulatory proteins. Our work demonstrates, for the first time, that both NHE1 and NBCn1 regulate cell cycle progression in breast cancer cells, and we propose that this involves cell cycle phase-specific pH_i regulation by the two transporters.     

A polarized localization of amino acid/carnitine transporter B(0,+) (ATB(0,+)) in the blood-brain barrier

Brain capillary endothelial cells control the uptake and efflux from the brain of many hydrophilic compounds due to highly specialized transporters often localized in a polarized way. Localization of Na⁺- and Cl⁻-dependent amino acid and carnitine transporter B^0,+ (ATB^0,+) was studied in a co-culture of bovine brain capillary endothelial cells (BBCEC) grown on filters above astrocytes (an in vitro blood-brain barrier model). Immunoblotting and three-dimensional immunocytochemistry analysis with anti-B^0,+antibodies demonstrated the presence of this transporter and its prevalent co-localization with P-glycoprotein i.e. at the apical side. The sensitivity of leucine uptake through the apical membrane to 2-aminobicyclo-[2.2.1]-heptane-2-carboxylic acid (BCH), d-serine as well as sodium and chloride replacement confirm the functioning of ATB^0,+ and suggests an important physiological role of ATB^0,+ in controlling the delivery of amino acids and carnitine to the brain.     

Insight into the molecular switch mechanism of human Rab5a from molecular dynamics simulations

Rab5a is currently a most interesting target because it is responsible for regulating the early endosome fusion in endocytosis and possibly the budding process. We utilized longtime-scale molecular dynamics simulations to investigate the internal motion of the wild-type Rab5a and its A30P mutant. It was observed that, after binding with GTP, the global flexibility of the two proteins is increasing, while the local flexibility in their sensitive sites (P-loop, switch I and II regions) is decreasing. Also, the mutation of Ala30 to Pro30 can cause notable flexibility variations in the sensitive sites. However, this kind of variations is dramatically reduced after binding with GTP. Such a remarkable feature is mainly caused by the water network rearrangements in the sensitive sites. These findings might be of use for revealing the profound mechanism of the displacements of Rab5a switch regions, as well as the mechanism of the GDP dissociation and GTP association.     

Structural basis for the voltage-gated Na+ channel selectivity of the scorpion alpha-like toxin BmK M1

Scorpion alpha-like toxins are proteins that act on mammalian and insect voltage-gated Na+ channels. Therefore, these toxins constitute an excellent target for examining the foundations that underlie their target specificity. With this motive we dissected the role of six critical amino acids located in the five-residue reverse turn (RT) and C-tail (CT) of the scorpion alpha-like toxin BmK M1. These residues were individually substituted resulting in 11 mutants and were subjected to a bioassay on mice, an electrophysiological characterization on three cloned voltage-gated Na+ channels (Nav1.2, Nav1.5 and para), a CD analysis and X-ray crystallography. The results reveal two molecular sites, a couplet of residues (8-9) in the RT and a hydrophobic surface consisting of residues 57 and 59-61 in the CT, where the substitution with specific residues can redirect the alpha-like characteristics of BmK M1 to either total insect or much higher mammal specificity. Crystal structures reveal that the pharmacological ramification of these mutants is accompanied by the reshaping of the 3D structure surrounding position 8. Furthermore, our results also reveal that residues 57 and 59-61, located at the CT, enclose the critical residue 58 in order to form a hydrophobic "gasket". Mutants of BmK M1 that interrupt this hydrophobic surface significantly gain insect selectivity.     

Analysis of the SLC4A1 gene in three Mexican patients with hereditary spherocytosis: Report of a novel mutation

We analyzed the SLC4A1 gene in three Mexican patients with Hereditary Spherocytosis (HS). The promoter and all 20 exons were investigated through heteroduplex analysis and DNA sequencing. No DNA changes were detected in one of the three patients. Two well-known polymorphisms, Memphis I and the Diego-a blood group, were detected in another one. In the third, the HS phenotype could be explained by the novel 1885_1888dupCCGG mutation found in heterozygosis. This frameshift mutation is predicted to result in a truncated and unstable protein lacking normal functions.     

Enhancement of gene detection frequencies by combining DNA-based stable-isotope probing with the construction of metagenomic DNA libraries

Summary DNA-based stable-isotope probing (SIP) using ¹³C-labeled growth substrates as bait is a powerful tool for the selective DNA isolation from microorganisms that are actively involved in consuming these substrates. To enhance the detection frequency of target genes in screens for new natural products, we have combined for the first time DNA-based SIP with the construction of metagenomic libraries. To isolate genes encoding coenzyme B₁₂-dependent glycerol dehydratases an enrichment of glycerol-fermenting microorganisms from a sediment sample of the Wadden Sea was performed by using glycerol–¹³C₃ as sole carbon source. Subsequently, the ¹³C-labeled DNA was separated from the naturally abundant ¹²C-DNA by density centrifugation, and used for library generation. Screening of the constructed libraries for the target genes revealed that the gene detection frequencies employing DNA-based SIP for enrichment of genomes harboring dehydratase genes were 2.1- to 3.8-fold higher than those recorded by using a traditional step with unlabeled glycerol for enrichment.     

Use of Chemically Modified Nucleotides to Determine a 62-Nucleotide RNA Crystal Structure: A Survey of Phosphorothioates,Br, Pt and Hg

Abstract

Two important challenges confronting RNA crystallographers are producing crystals and finding isomorphous heavy-atom derivatives. Non-isomorphism can be addressed by determining the phases using the multiwavelength anomalous dispersion (MAD) method. These phases can be greatly improved by combining phases from MAD experiments done on different heavy-atom derivatives. Heavy-atom derivatives can be created by chemically modifying the RNA through covalent attachment of bromine or mercury to C5 of pyrimidines or [Pt(NH₃)₃]²⁺ to N7 of guanine. While phosphorothioates can provide mercury binding sites, disorder can reduce their value for phase determination. The location of these chemical modifications is critical since crystallization of these derivatized RNAs is sensitive to heavy atom induced conformational alterations and crystal packing.     

Towards an understanding of the molecular basis of plants K+ transport: Characterization of cloned K+ transport cDNAs

Recently, two K⁺-transport cDNAs, KAT1 and AKT1, were cloned in Arabidopsis thaliana. These cDNAs had structural similarities to K⁺ channel genes in animals, and also conferred the ability for growth on micromolar levels of K⁺ when expressed in K⁺ transport-defective yeast mutants. In this study, we examined the possibility that KAT1 encodes the high-affinity K⁺ transport system that has been previously characterized in plant roots, by studying the concentration-dependent kinetics of K⁺ transport for KAT1 expressed in Xenopus oocytes and Saccharomyces cerevisiae. In both organisms, the K⁺ transport system encoded by KAT1 yielded Michaelis-Menten kinetics with a high K_m for K⁺ (35 mM in oocytes, 0.6 mM in yeast cells). Furthermore, Northern analysis indicated that KAT1 is expressed primarily in the Arabidopsis shoot. These results strongly suggest that the system encoded by KAT1 is not a root high-affinity K⁺ transporter.     

The role of cattle in the volatile loss of nitrogen from a shortgrass steppe

The cycling and volatile loss of N derived from cattle urine at upland and lowland sites within the shortgrass steppe of eastern Colorado was studied, using¹⁵N-labelled urea as an N source. Losses of NH ₀ ³ were determined by direct measurement and by difference. Losses were higher from coarse (27% summer, 12% winter) than from fine textured (0–2%) soils. Immobilization and plant uptake of N accounted for significant amounts of added N. Extrapolating our plot measurements to a typical pasture, using spatially and temporally stratified urine deposition data, losses from upland sites were calculated to be 0.016 g N · m^-2 · y^-1, while losses from lowland sites were negligible. This resulted in an average loss of 0.011 g N · m^-2 · y^-1 for a pasture divided 70:30 between uplands and lowlands. The loss of urine N calculated assuming no spatial stratification would be sevenfold higher (0.076 g N · m^-2 · y^-1). Losses of NH ₀ ³ from urine, animal biomass removal, and NH₂O loss totaled only 0.07 g N · m^-2 · y^-1 , or about 25% of wet deposition input. We calculated a potential loss of NH ₀ ³ from senescing vegetation of 0.26 g N · m^-2 · y^-1, an order of magnitude larger than all other losses combined.     

The protein kinase ERK3 is encoded by a single functional gene: genomic analysis of the ERK3 gene family

Extracellular signal-regulated kinase 3 (ERK3) is a distantly related member of the mitogen-activated protein (MAP) kinase family of serine/threonine kinases. Here, we report the characterization of the genomic loci encoding ERK3 in mice and humans. The mouse ERK3 gene (Mapk6) spans more than 20 kb and is split into six exons. Its structure is similar to that of the human MAPK6 gene, which extends over 40 kb. We also identified and characterized a mouse Mapk6 processed pseudogene. In humans, database analysis has revealed the presence of six MAPK6 processed pseudogenes localized on four different chromosomes. We further show that the structure of MAPK6 is closely related to that of the gene encoding the homologous protein kinase p63(MAPK) (MAPK4), suggesting that the two genes arose by duplication. Our analysis demonstrates that the ERK3 subfamily of MAP kinase genes is composed of two functional genes, MAPK6 and MAPK4, and several pseudogenes.     

On the molecular basis of ion permeation in the epithelial Na+ channel.

The epithelial Na+ channel (ENaC) is highly selective for Na+ and Li+ over K+ and is blocked by the diuretic amiloride. ENaC is a heterotetramer made of two alpha, one beta, and one gamma homologous subunits, each subunit comprising two transmembrane segments. Amino acid residues involved in binding of the pore blocker amiloride are located in the pre-M2 segment of beta and gamma subunits, which precedes the second putative transmembrane alpha helix (M2). A residue in the alpha subunit (alphaS589) at the NH2 terminus of M2 is critical for the molecular sieving properties of ENaC. ENaC is more permeable to Li+ than Na+ ions. The concentration of half-maximal unitary conductance is 38 mM for Na+ and 118 mM for Li+, a kinetic property that can account for the differences in Li+ and Na+ permeability. We show here that mutation of amino acid residues at homologous positions in the pre-M2 segment of alpha, beta, and gamma subunits (alphaG587, betaG529, gammaS541) decreases the Li+/Na+ selectivity by changing the apparent channel affinity for Li+ and Na+. Fitting single-channel data of the Li+ permeation to a discrete-state model including three barriers and two binding sites revealed that these mutations increased the energy needed for the translocation of Li+ from an outer ion binding site through the selectivity filter. Mutation of betaG529 to Ser, Cys, or Asp made ENaC partially permeable to K+ and larger ions, similar to the previously reported alphaS589 mutations. We conclude that the residues alphaG587 to alphaS589 and homologous residues in the beta and gamma subunits form the selectivity filter, which tightly accommodates Na+ and Li+ ions and excludes larger ions like K+.     

The structure of the Na+,K+-ATPase and mapping of isoform differences and disease-related mutations

The Na+,K+-ATPase transforms the energy of ATP to the maintenance of steep electrochemical gradients for sodium and potassium across the plasma membrane. This activity is tissue specific, in particular due to variations in the expressions of the alpha subunit isoforms one through four. Several mutations in alpha2 and 3 have been identified that link the specific function of the Na+,K+-ATPase to the pathophysiology of neurological diseases such as rapid-onset dystonia parkinsonism and familial hemiplegic migraine type 2. We show a mapping of the isoform differences and the disease-related mutations on the recently determined crystal structure of the pig renal Na+,K+-ATPase and a structural comparison to Ca2+-ATPase. Furthermore, we present new experimental data that address the role of a stretch of three conserved arginines near the C-terminus of the alpha subunit (Arg1003-Arg1005).     

The pur6 gene of the puromycin biosynthetic gene cluster from Streptomyces alboniger encodes a tyrosinyl-aminonucleoside synthetase

The pur6 gene of the puromycin biosynthetic gene (pur) cluster from Streptomyces alboniger is shown to be essential for puromycin biosynthesis. Cell lysates from this mycelial bacterium were active in linking L-tyrosine to both 3'-amino-3'-deoxyadenosine and N6,N6-dimethyl-3'-amino-3'-deoxyadenosine with a peptide-like bond. Identical reactions were performed by cell lysates from Streptomyces lividans or Escherichia coli transformants that expressed pur6 from a variety of plasmid constructs. Physicochemical and biochemical analyses suggested that their products were tridemethyl puromycin and O-demethylpuromycin, respectively. Therefore, it appears that Pur6 is the tyrosinyl-aminonucleoside synthetase of the puromycin biosynthetic pathway.     

The role of ion antiporters in the maintenance of intracellular pH in rat vascular smooth muscle cells

Vascular smooth muscle intracellular pH is maintained by the Na⁺/H⁺ and Cl^–/HCO ₃ ^– antiporters. The Na⁺/H⁺ exchanger is a major route of H⁺ extrusion in most eukaryotic cells and is present in vascular smooth muscle cells in a similar capacity. It extrudes H^– into the extracellular space in exchange for Na⁺. The Cl^–/HCO ₃ ^– exchanger plays an analogous role to lower the pH of vascular smooth muscle cells when increases in intracellular pH occur. Its activity has also been demonstrated in A7r5 and A10 vascular smooth muscle cells. The Na⁺/H⁺ exchanger is regulated by a number of agents which act through inositol trisphosphate/diacylglycerol, to stimulate the antiporter. Calcium-calmodulin dependent protein kinase may also activate the antiporter in vivo. Phosphorylation of the Cl^–/HCO ₃ ^– exchanger has also been observed but its physiological role is not known. Both these antiporters exist in the plasma membrane as integral proteins with free acidic cytoplasmic termini. These regions may be important in sensing changes in intracellular pH, to which these antiporters respond.Abbreviations CaM Calmodulin - DCCD Dicylohexyl-Carbodiimide - DG Diacylglycerol - DIDS-4 4-Diisthiocyanostilbene-2,2-Disulfonic Acid - IP₃ Inositol Trisphosphate - PKC protein Kinase C - SITS-4 4-Acetamido-4-Isothiocyanstilbene-2,2-Disulfonate - VSMC Vascular Smooth Muscle Cell     

The role of various calcium and potassium channels in the regulation of somatodendritic serotonin release

We prepared slices from midbrain containing the raphe nuclei and from hippocampus of rats. The brain slices were loaded with [³H]serotonin and superfused in order to measure the release of radioactivity at rest and in response to electrical stimulation. No difference was observed in the resting and stimulated fractional release of tritium in the somatodendritic and axon terminal parts of serotonergic neurons. The selective 5-HT_1A receptor agonist 8-OH-DPAT decreased the electrically induced tritium effux from raphe nuclei slices preloaded with [³H]serotonin, and this inhibition was reversed by 5-HT_1A receptor antagonist (+)WAY-100135. The 5-HT_1B receptor agonist CGS-12066B but not 8-OH-DPAT, inhibited the stimulation-evoked tritium efflux from hippocampal slices after labeling with [³H]serotonin. The electrical stimulation-evoked tritium efflux in raphe nuclei slices incubate with [³H]serotonin was completely external Ca²⁺-dependent, and omega-conotoxin GVIA and Cd²⁺, but not diltiazem, inhibited the tritium overflow. In raphe nuclei slices 4-aminopyridine enhanced the electrical stimulation-induced trititum release in a concentration-dependent manner. The inhibition of tritium efflux by 8-OH-DPAT was abolished with 4-aminopyridine. Glibenclamide or tolbutamide proved to be ineffective. These data indicate that (1) different 5-HT receptor subtypes (5-HT_1A and 5-HT_1B) regulate dendritic and axon terminal 5-HT release; (2) serotonin release from the dendrites may be regulated by the voltage-sensitive N-type Ca²⁺ channels; (3) the 5-HT_1A receptor-mediated inhibition of serotonin release may be due to opening of voltage-sensitive K⁺ channels.