Modulation of drug-stimulated ATPase activity of human MDR1/P-glycoprotein by cholesterol

MDR1 (multidrug resistance 1)/P-glycoprotein is an ATP-driven transporter which excretes a wide variety of structurally unrelated hydrophobic compounds from cells. It is suggested that drugs bind to MDR1 directly from the lipid bilayer and that cholesterol in the bilayer also interacts with MDR1. However, the effects of cholesterol on drug-MDR1 interactions are still unclear. To examine these effects, human MDR1 was expressed in insect cells and purified. The purified MDR1 protein was reconstituted in proteoliposomes containing various concentrations of cholesterol and enzymatic parameters of drug-stimulated ATPase were compared. Cholesterol directly binds to purified MDR1 in a detergent soluble form and the effects of cholesterol on drug-stimulated ATPase activity differ from one drug to another. The effects of cholesterol on K(m) values of drug-stimulated ATPase activity were strongly correlated with the molecular mass of that drug. Cholesterol increases the binding affinity of small drugs (molecular mass <500 Da), but does not affect that of drugs with a molecular mass of between 800 and 900 Da, and suppresses that of valinomycin (molecular mass >1000 Da). V(max) values for rhodamine B and paclitaxel are also increased by cholesterol, suggesting that cholesterol affects turnover as well as drug binding. Paclitaxel-stimulated ATPase activity of MDR1 is enhanced in the presence of stigmasterol, sitosterol and campesterol, as well as cholesterol, but not ergosterol. These results suggest that the drug-binding site of MDR1 may best fit drugs with a molecular mass of between 800 and 900 Da, and that cholesterol may support the recognition of smaller drugs by adjusting the drug-binding site and play an important role in the function of MDR1.     

Genome-wide screening of genes required for swarming motility in Escherichia coli K-12

Escherichia coli K-12 has the ability to migrate on semisolid media by means of swarming motility. A systematic and comprehensive collection of gene-disrupted E. coli K-12 mutants (the Keio collection) was used to identify the genes involved in the swarming motility of this bacterium. Of the 3,985 nonessential gene mutants, 294 were found to exhibit a strongly repressed-swarming phenotype. Further, 216 of the 294 mutants displayed no significant defects in swimming motility; therefore, the 216 genes were considered to be specifically associated with the swarming phenotype. The swarming-associated genes were classified into various functional categories, indicating that swarming is a specialized form of motility that requires a wide variety of cellular activities. These genes include genes for tricarboxylic acid cycle and glucose metabolism, iron acquisition, chaperones and protein-folding catalysts, signal transduction, and biosynthesis of cell surface components, such as lipopolysaccharide, the enterobacterial common antigen, and type 1 fimbriae. Lipopolysaccharide and the enterobacterial common antigen may be important surface-acting components that contribute to the reduction of surface tension, thereby facilitating the swarm migration in the E. coli K-12 strain.     

Removal and recovery of phosphorous from swine wastewater by demonstration crystallization reactor and struvite accumulation device

A demonstration crystallization reactor and struvite accumulation device for the removal and recovery of phosphorous was constructed and their performance was evaluated using actual swine wastewater for 3.5 years. The wastewater pH was increased by aeration, and the concentrations of total P and soluble PO(4)-P were reduced by a struvite crystallization reaction induced under a high pH condition. A 30% MgCl(2) addition was effective in enhancing the struvite crystallization reaction. The concentrations of suspended solids, total Zn and total Cu, were also decreased by the settling function of the reactor. On removing the efficiencies of these components, no noticeable seasonal fluctuation in performance was observed during the 3.5-year operation. In terms of maximum yield, 171g struvite was obtained from 1m(3) swine wastewater by the demonstration accumulation device for struvite recovery. The recovered struvite needed only air-drying before use since it was approximately 95% pure even without washing.     

Blocking neddylation elicits antiviral effect against hepatitis B virus replication

Molecular Biology Reports - Hepatitis B Virus (HBV) is the most common cause of chronic liver disease worldwide. The mechanisms that regulate HBV viral replication remain poorly defined. Here, we...     

Impaired hippocampal long-term potentiation and failure of learning in β1,4-N-acetylgalactosaminyltransferase gene transgenic mice

Gangliosides (sialic acid-containing glycosphingolipids) play important roles in many physiological functions, including synaptic plasticity in the hippocampus, which is considered as a cellular mechanism of learning and memory. In the present study, three types of synaptic plasticity, long-term potentiation (LTP), long-term depression (LTD) and reversal of LTP (depotentiation, DP), in the field excitatory post-synaptic potential in CA1 hippocampal neurons and learning behavior were examined in β1,4-N-acetylgalactosaminyltransferase (β1,4 GalNAc-T; GM2/GD2 synthase) gene transgenic (TG) mice, which showed a marked decrease in b-pathway gangliosides (GQ1b, GT1b and GD1b) in the brain and isolated hippocampus compared with wild-type (WT) mice. The magnitude of the LTP induced by tetanus (100 pulses at 100?Hz) in TG mice was significantly smaller than that in control WT mice, whereas there was no difference in the magnitude of the LTD induced by three short trains of low-frequency stimulation (LFS) (200 pulses at 1?Hz) at 20?min intervals between the two groups of mice. The reduction in the LTP produced by delivering three trains of LFS (200 pulses at 1?Hz, 20?min intervals) was significantly greater in the TG mice than in the WT mice. Learning was impaired in the four-pellet taking test (4PTT) in TG mice, with no significant difference in daily activity or activity during the 4PTT between TG and WT mice. These results suggest that the overexpression of β1,4 GalNAc-T resulted in altered synaptic plasticity of LTP and DP in hippocampal CA1 neurons and learning in the 4PTT, and this is attributable to the shift from b-pathway gangliosides to a-pathway gangliosides.     

Effects of struvite formation and nitratation promotion on nitrogenous emissions such as NH3, N2O and NO during swine manure composting

To reduce nitrogenous emissions from composting, two different countermeasures were applied simultaneously in swine manure composting. One was forming struvite by adding Mg and P at the start of composting, and the other was to promote nitratation (nitrite being oxidized nitrate) by adding nitrite-oxidizing bacteria after the thermophilic phase of composting. In the laboratory- and mid-scale composting experiments, 25-43% of NH3, 52-80% of N2O and 96-99% of NO emissions were reduced. From the nitrogen balance, it was revealed that the struvite formation reduced not only NH3, but also other nitrogenous emissions except N2O. The amount of total nitrogen losses was reduced by 60% by the two combined countermeasures, against 51% by the struvite formation alone. However, the nitratation promotion dissolved struvite crystals due to the pH decline, diminishing the effect of struvite as a slow-release fertilizer.     

Investigation by imaging mass spectrometry of biomarker candidates for aging in the hair cortex

Background

Human hair is one of the essential components that define appearance and is a useful source of samples for non-invasive biomonitoring. We describe a novel application of imaging mass spectrometry (IMS) of hair biomolecules for advanced molecular characterization and a better understanding of hair aging. As a cosmetic and biomedical application, molecules whose levels in hair altered with aging were comprehensively investigated.

Methods

Human hair was collected from 15 young (20±5 years old) and 15 older (50±5 years old) volunteers. Matrix-free laser desorption/ionization IMS was used to visualize molecular distribution in the hair sections. Hair-specific ions displaying a significant difference in the intensities between the 2 age groups were extracted as candidate markers for aging. Tissue localization of the molecules and alterations in their levels in the cortex and medulla in the young and old groups were determined.

Results

Among the 31 molecules detected specifically in hair sections, 2—one at m/z 153.00, tentatively assigned to be dihydrouracil, and the other at m/z 207.04, identified to be 3,4-dihydroxymandelic acid (DHMA)—exhibited a higher signal intensity in the young group than in the old, and 1 molecule at m/z 164.00, presumed to be O-phosphoethanolamine, displayed a higher intensity in the old group. Among the 3, putative O-phosphoethanolamine showed a cortex-specific distribution. The 3 molecules in cortex presented the same pattern of alteration in signal intensity with aging, whereas those in medulla did not exhibit significant alteration.

Conclusion

Three molecules whose levels in hair altered with age were extracted. While they are all possible markers for aging, putative dihydrouracil and DHMA, are also suspected to play a role in maintaining hair properties and could be targets for cosmetic supplementation. Mapping of ion localization in hair by IMS is a powerful method to extract biomolecules in specified regions and determine their tissue distribution.     

Purification and cDNA cloning of chloroplastic monodehydroascorbate reductase from spinach

The chloroplastic isoform of monodehydroascorbate (MDA) radical reductase was purified from spinach chloroplasts and leaves. The cDNA of chloroplastic MDA reductase was cloned, and its deduced amino acid sequence, consisting of 497 residues, showed high homology with those of putative organellar MDA reductases deduced from cDNAs of several plants. The amino acid sequence of the amino terminal of the purified enzyme suggested that the chloroplastic enzyme has a transit peptide consisting of 53 residues. A southern blot analysis suggested the occurrence of a gene encoding another isoform homologous to the chloroplastic isoform in spinach. The recombinant enzyme was highly expressed in Eschericia coli using the cDNA, and purified to a homogeneous state with high specific activity. The enzyme properties of the chloroplastic isoform are presented in comparison with those of the cytosolic form.