Biochemical characterization of human ZIP13 protein: a homo-dimerized zinc transporter involved in the spondylocheiro dysplastic Ehlers-Danlos syndrome

The human SLC39A13 gene encodes ZIP13, a member of the LZT (LIV-1 subfamily of ZIP zinc transporters) family. The ZIP13 protein is important for connective tissue development, and its loss of function is causative for the spondylocheiro dysplastic form of Ehlers-Danlos syndrome. However, this protein has not been characterized in detail. Here we report the first detailed biochemical characterization of the human ZIP13 protein using its ectopic expressed and the purified recombinant protein. Protease accessibility, microscopic, and computational analyses demonstrated that ZIP13 contains eight putative transmembrane domains and a unique hydrophilic region and that it resides with both its N and C termini facing the luminal side on the Golgi. Analyses including cross-linking, immunoprecipitation, Blue Native-PAGE, and size-exclusion chromatography experiments indicated that the ZIP13 protein may form a homo-dimer. We also demonstrated that ZIP13 mediates zinc influx, as assessed by monitoring the expression of the metallothionein gene and by detecting the intracellular zinc level with a zinc indicator, FluoZin-3. Our data indicate that ZIP13 is a homo-dimerized zinc transporter that possesses some domains that are not found in other LZT family members. This is the first biochemical characterization of the physiologically important protein ZIP13 and the demonstration of homo-dimerization for a mammalian ZIP zinc transporter family member. This biochemical characterization of the human ZIP13 protein provides important information for further investigations of its structural characteristics and function.     

Synthesis and evaluation of 6-[1-(2-[(18)F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline for positron emission tomography imaging of the metabotropic glutamate receptor type 1 in brain

The purpose of this study was to synthesize 6-[1-(2-[¹⁸F]fluoro-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline ([¹⁸F]FPTQ, [¹⁸F]7a) and to evaluate its potential as a positron emission tomography ligand for imaging metabotropic glutamate receptor type 1 (mGluR1) in the rat brain. Compound [¹⁸F]7a was synthesized by [¹⁸F]fluorination of 6-[1-(2-bromo-3-pyridyl)-5-methyl-1H-1,2,3-triazol-4-yl]quinoline (7b) with potassium [¹⁸F]fluoride. At the end of synthesis, 1280-1830 MBq (n = 8) of [¹⁸F]7a was obtained with >98% radiochemical purity and 118-237 GBq/??mol specific activity using 3300-4000 MBq of [¹⁸F]F⁻. In vitro autoradiography showed that [¹⁸F]7a had high specific binding with mGluR1 in the rat brain. Biodistribution study using a dissection method and small-animal PET showed that [¹⁸F]7a had high uptake in the rat brain. The uptake of radioactivity in the cerebellum was reduced by unlabeled 7a and mGluR1-selective ligand JNJ-16259685 (2), indicating that [¹⁸F]7a had in vivo specific binding with mGluR1. Because of a low amount of radiolabeled metabolite present in the brain, [¹⁸F]7a may have a limiting potential for the in vivo imaging of mGluR1 by PET.     

Improved performance by replacing iminodiacetic residues with glyceryl residues in symmetrically branched oligoglycerols

Synthesis of a symmetrically branched diglycerol (BGL002, involving one iminodiacetic residue) as a G2 dendron, and the tetradecaglycerol (BGL014, involving one iminodiacetic residue) as a G4 dendron, is described. Several members of the BGL family of G2-G4 dendrons were assembled, with G2 bearing four hydroxyl groups at the terminus region, G3 bearing eight, and G4 bearing sixteen. It is noteworthy that triglycerol (BGL003, including no iminodiacetic residue), has a water-solubility ten times higher than BGL002, and the liposome surrounded by BGL014 has a duration period in blood vessel roughly two times longer than the liposome surrounded by dodecaglycerol (BGL012, including three iminodiacetic residues).     

Differentiation,polarization, and migration of human induced pluripotent stem cell-derived neural progenitor cells co-cultured with a human glial cell line with radial glial-like characteristics

Here we established a unique human glial cell line, GDC90, derived from a human glioma and demonstrated its utility as a glial scaffold for the polarization and differentiation of human induced pluripotent stem cell-derived neural progenitor cells (iPSC-NPCs). When co-cultured with GDC90 cells, iPSC-NPCs underwent rapid polarization and neurite extension along the radially spreading processes of the GDC90 cells, and showed migratory behavior. This method is potentially useful for detailed examination of neurites or for controlling neurites behavior for regenerative medicine.     

A study on cryoprotectant solution suitable for vitrification of rat two-cell stage embryos

The present study was performed to develop a suitable cryoprotectant solution for cryopreservation of rat two-cell stage embryos. First, we examined the cell permeability of several cryoprotectants; propylene glycol had the fastest permeability compared to dimethyl sulfoxide, ethylene glycol, and glycerol. Embryos were then exposed to a solution containing propylene glycol to evaluate its effects on fetal development. As the development was similar to that of fresh embryos, P10 (10% v/v propylene glycol in PB1) was used as a pretreatment solution. Next, the effects of the vitrification solution components (sucrose, propylene glycol, ethylene glycol, and Percoll) were examined by observing the vitrification status; 10% v/v propylene glycol, 30% v/v ethylene glycol, 0.3 mol sucrose, and 20% v/v Percoll in PB1 (PEPeS) was the minimum essential concentration for effective vitrification without the formation of ice crystals or freeze fractures.     

Carnosol,rosemary ingredient,induces apoptosis in adult T-cell leukemia/lymphoma cells via glutathione depletion: proteomic approach using fluorescent two-dimensional differential gel electrophoresis

Adult T-cell leukemia/lymphoma (ATL) is a fatal malignancy caused by infection with human T-lymphotropic virus type-1 and there is no accepted curative therapy for ATL. We searched for biological active substances for the prevention and treatment of ATL from several species of herbs. The ATL cell growth-inhibitory activity and apoptosis assay showed that carnosol, which is an ingredient contained in rosemary (Rosmarinus officinalis), induced apoptosis in ATL cells. Next, to investigate the apoptosis-inducing mechanism of carnosol, we applied proteomic analysis using fluorescent two-dimensional differential gel electrophoresis and mass spectrometry. The proteomic analysis showed that the expression of reductases, enzymes in glycolytic pathway, and enzymes in pentose phosphate pathway was increased in carnosol-treated cells, compared with untreated cells. These results suggested that carnosol affected the redox status in the cells. Further, the quantitative analysis of glutathione, which plays the central role for the maintenance of intracellular redox status, indicated that carnosol caused the decrease of glutathione in the cells. Further, N-acetyl-l-cystein, which is precursor of glutathione, canceled the efficiency of carnosol. From these results, it was suggested that the apoptosis-inducing activity of carnosol in ATL cells was caused by the depletion of glutathione.     

The role of wetland microinvertebrates in spreading human diseases

The increasing loads of anthropogenic pollutants, compounded with climate change events, are likely to induce environmental changes in many wetlands with impacts on the native microinvertebrates and pathogens causing increased occurrence of water-borne diseases, which affect millions of people each year. In wetlands bacterial pathogens are actively preyed on by many protozoa and filter-feeding organisms but this predation can be compensated by the nourishment and protection offered by certain microinvertebrates, acting as hosts, e.g., chitinous rotifers, copepods and cladocerans. The complex interactions of ecological, biological, and genetic components mediate disease-causing organisms to exploit microinvertebrate hosts to occupy diverse niches, obtain nutrition, and withstand physico-chemical stresses. The persistence of the human pathogens in wetlands is often enabled by their association with microinvertebrates and also depends on their quorum sensing mediated colonization, biofilm formation, switching into dormant stage, and horizontal transfer of adaptive genes. The symbiosis with microinvertebrates is facilitated by the pathogen’s immune evasion and fitness factors, e.g., Type-IV pili, capsular-polysaccharides, nutrient transportation, virulence and binding proteins, proteases, chitinases, and secretion systems. Spatio-temporal variation in the population of copepods and aquatic eggs/larvae of mosquitoes and midge flies, which act as vectors, can influence the outbreaks of cholera, diarrhea, malaria, dengue, filariasis and drucunculiasis. Changes in climatic factors (temperature, salinity, cyclones, rainfall, etc.) and anthropogenic pollutions (sewage, fertilizer and insecticide) may modify the abundance and biodiversity of microinvertebrates, and thus possibly exacerbate the persistence and dispersal of water-borne pathogens. Thus there is a need to adopt ecohydrological and eco-friendly interventions for managing wetlands while conserving them.     

Simulation and estimation of gene number in a biological pathway using almost complete saturation mutagenesis screening of haploid mouse cells

Background

Genome-wide saturation mutagenesis and subsequent phenotype-driven screening has been central to a comprehensive understanding of complex biological processes in classical model organisms such as flies, nematodes, and plants. The degree of “saturation” (i.e., the fraction of possible target genes identified) has been shown to be a critical parameter in determining all relevant genes involved in a biological function, without prior knowledge of their products. In mammalian model systems, however, the relatively large scale and labor intensity of experiments have hampered the achievement of actual saturation mutagenesis, especially for recessive traits that require biallelic mutations to manifest detectable phenotypes.

Results

By exploiting the recently established haploid mouse embryonic stem cells (ESCs), we present an implementation of almost complete saturation mutagenesis in a mammalian system. The haploid ESCs were mutagenized with the chemical mutagen N-ethyl-N-nitrosourea (ENU) and processed for the screening of mutants defective in various steps of the glycosylphosphatidylinositol-anchor biosynthetic pathway. The resulting 114 independent mutant clones were characterized by a functional complementation assay, and were shown to be defective in any of 20 genes among all 22 known genes essential for this well-characterized pathway. Ten mutants were further validated by whole-exome sequencing. The predominant generation of single-nucleotide substitutions by ENU resulted in a gene mutation rate proportional to the length of the coding sequence, which facilitated the experimental design of saturation mutagenesis screening with the aid of computational simulation.

Conclusions

Our study enables mammalian saturation mutagenesis to become a realistic proposition. Computational simulation, combined with a pilot mutagenesis experiment, could serve as a tool for the estimation of the number of genes essential for biological processes such as drug target pathways when a positive selection of mutants is available.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-1016) contains supplementary material, which is available to authorized users.