Abstract
Neurological disorders affect millions of people worldwide and appear to be on the rise. Whereas the reason for this increase remains unknown, environmental factors are a suspected ...contributor. Hence, there is an urgent need to develop more complex, biologically relevant, and predictive in vitro assays to screen larger sets of compounds with the potential for neurotoxicity. Here, we employed a human induced pluripotent stem cell (iPSC)-based 3D neural platform composed of mature cortical neurons and astrocytes as a model for this purpose. The iPSC-derived human 3D cortical neuron/astrocyte co-cultures (3D neural cultures) present spontaneous synchronized, readily detectable calcium oscillations. This advanced neural platform was optimized for high-throughput screening in 384-well plates and displays highly consistent, functional performance across different wells and plates. Characterization of oscillation profiles in 3D neural cultures was performed through multi-parametric analysis that included the calcium oscillation rate and peak width, amplitude, and waveform irregularities. Cellular and mitochondrial toxicity were assessed by high-content imaging. For assay characterization, we used a set of neuromodulators with known mechanisms of action. We then explored the neurotoxic profile of a library of 87 compounds that included pharmaceutical drugs, pesticides, flame retardants, and other chemicals. Our results demonstrated that 57% of the tested compounds exhibited effects in the assay. The compounds were then ranked according to their effective concentrations based on in vitro activity. Our results show that a human iPSC-derived 3D neural culture assay platform is a promising biologically relevant tool to assess the neurotoxic potential of drugs and environmental toxicants.
Development of more complex, biologically relevant, and predictive cell-based assays for compound screening is a major challenge in drug discovery. The focus of this study was to establish ...high-throughput compatible three-dimensional (3D) cardiotoxicity assays using human induced pluripotent stem cell-derived cardiomyocytes. Using both high-content imaging and fast kinetic fluorescence imaging, the impact of various compounds on the beating rates and patterns of cardiac spheroids was monitored by changes in intracellular Ca
levels with calcium-sensitive dyes. Advanced image analysis methods were implemented to provide multiparametric characterization of the Ca
oscillation patterns. In addition, we used confocal imaging and 3D analysis methods to characterize compound effects on the morphology of 3D spheroids. This phenotypic assay allows for the characterization of parameters such as beating frequency, amplitude, peak width, rise and decay times, as well as cell viability and morphological characteristics. A set of 22 compounds, including a number of known cardioactive and cardiotoxic drugs, was assayed at different time points, and the calculated EC
values for compound effects were compared between 3D and two-dimensional (2D) model systems. A significant concordance in the phenotypes was observed for compound effects between the two models, but essential differences in the concentration responses and time dependencies of the compound-induced effects were observed. Together, these results indicate that 3D cardiac spheroids constitute a functionally distinct biological model system from traditional flat 2D cultures. In conclusion, we have demonstrated that phenotypic assays using 3D model systems are enabled for screening and suitable for cardiotoxicity assessment in vitro.
Cotransport of Water by the Na+/glucose Cotransporter Donald D. F. Loo; Zeuthen, Thomas; Chandy, Grischa ...
Proceedings of the National Academy of Sciences - PNAS,
11/1996, Letnik:
93, Številka:
23
Journal Article
Recenzirano
Odprti dostop
Water is transported across epithelial membranes in the absence of any hydrostatic or osmotic gradients. A prime example is the small intestine, where 10 liters of water are absorbed each day. ...Although water absorption is secondary to active solute transport, the coupling mechanism between solute and water flow is not understood. We have tested the hypothesis that water transport is directly linked to solute transport by cotransport proteins such as the brush border Na+/glucose cotransporter. The Na+/glucose cotransporter was expressed in Xenopus oocytes, and the changes in cell volume were measured under sugar-transporting and nontransporting conditions. We demonstrate that 260 water molecules are directly coupled to each sugar molecule transported and estimate that in the human intestine this accounts for 5 liters of water absorption per day. Other animal and plant cotransporters such as the Na+/Cl-/γ -aminobutyric acid, Na+/iodide and H+/amino acid transporters are also able to transport water and this suggests that cotransporters play an important role in water homeostasis.
The rabbit Na + -glucose (SGLT1) and the human Na + -Cl â -GABA (GAT1) cotransporters were expressed in Xenopus laevis oocytes, and passive Na + and water transport were studied using electrical ...and optical techniques. Passive water permeabilities ( L p ) of the cotransporters were determined from the changes in oocyte volume in response to osmotic gradients. The specific SGLT1
and GAT1 L p values were obtained by measuring L p in the presence and absence of blockers (phlorizin and SKF89976A). In the presence of the blockers, the L p values of oocytes expressing SGLT1 and GAT1 were indistinguishable from the L p of control oocytes. Passive Na + transport (Na + leak) was obtained from the blocker-sensitive Na + currents in the absence of substrates (glucose and GABA).
Passive Na + and water transport through SGLT1 were blocked by phlorizin with the same sensitivity (inhibitory constant ( K i ), 3-5 μM). When Na + was replaced with Li + , phlorizin also inhibited Li + and water transport, but with a lower affinity ( K i , 100 μM). When Na + was replaced by choline, which is not transported, the SGLT1 L p was indistinguishable from that in Na + or Li + , but in this case water transport was less sensitive to phlorizin.
The activation energies ( E a ) for passive Na + and water transport through SGLT1 were 21 and 5 kcal mol â1 , respectively. The high E a for Na + transport is comparable to that of Na + -glucose cotransport and indicates that the process is dependent on conformational changes of the protein, while the low E a for water transport is similar to that of water channels (aquaporins).
GAT1 also behaved as an SKF89976A-sensitive water channel. We did not observe passive Na + transport through GAT1.
We conclude that passive water and Na + transport through cotransporters depend on different mechanisms: Na + transport occurs by a saturable uniport mechanism, and water permeation is through a low conductance water channel. In the
case of SGLT1, we suggest that both the water channel and water cotransport could contribute to isotonic fluid transport across
the intestinal brush border membrane.
Upon infection of soybean roots, nitrogen-fixing bacteria become enclosed in a specific organelle known as the symbiosome. The symbiosome membrane (SM) is a selectively permeable barrier that ...controls metabolite flux between the plant cytosol and the symbiotic bacterium inside. Nodulin 26 (NOD 26), a member of the aquaporin (AQP) water channel family, is a major protein component of the SM. Expression of NOD 26 in Xenopus oocytes gave a mercury-sensitive increase in osmotic water permeability (Pf). To define the biophysical properties of NOD 26 water channels in their native membranes, symbiosomes were isolated from soybean root nodules and the SM separated as vesicles from the bacteria. Permeabilities were measured using stopped-flow fluorimetry in SM vesicles with entrapped carboxyfluorescein. Osmotic water permeability (Pf) of SM was high, with a value of 0.05 ± 0.003 cm/s observed at 20 °C (mean ± S.E.; n = 15). Water flow exhibited a low activation energy, was inhibited by HgCl2 (0.1 mm), and exhibited a unit conductance of 3.2 ± 1.3 × 10−15 cm3/s, a value 30-fold lower than that of AQP 1, the red blood cell water channel. Diffusive water permeability (Pd) was 0.0024 ± 0.0002 cm/s, and the resulting Pf toPd ratio was 18.3, indicating that water crosses the SM in single file fashion via the NOD 26 water channel. In addition to high water permeability, SM vesicles also show high mercury-sensitive permeability to glycerol and formamide, but not urea, suggesting that NOD 26 also fluxes these solutes. Overall, we conclude that NOD 26 acts as a water channel with a single channel conductance that is 30-fold lower than AQP 1. Because the solutes that permeate NOD 26 are far larger than water, and water appears to cross the channel via a single file pathway, solute flux across NOD 26 appears to occur by a pathway that is distinct from that for water.
1 Department of Molecular and Cell Biology and
2 Department of Physics, University of California, Berkeley,
California 94720-3200
Work addressing whether cystic fibrosis
transmembrane conductance ...regulator (CFTR) plays a role in regulating
organelle pH has remained inconclusive. We engineered a pH-sensitive
excitation ratiometric green fluorescent protein (pHERP) and targeted
it to the Golgi with sialyltransferase (ST). As determined by
ratiometric imaging of cells expressing ST-pHERP, Golgi pH
(pH G ) of HeLa cells was 6.4, while pH G of
mutant ( F508) and wild-type CFTR-expressing (WT-CFTR) respiratory
epithelia were 6.7-7.0. Comparison of genetically matched F508
and WT-CFTR cells showed that the absence of CFTR statistically
increased Golgi acidity by 0.2 pH units, though this small difference
was unlikely to be physiologically important. Golgi pH was maintained
by a H + vacuolar (V)-ATPase countered by a H +
leak, which was unaffected by CFTR. To estimate Golgi proton permeability ( P H + ), we modeled
transient changes in pH G induced by inhibiting the V-ATPase
and by acidifying the cytosol. This analysis required knowing Golgi
buffer capacity, which was pH dependent. Our in vivo estimate is that
Golgi P H + = 7.5 × 10 4 cm/s when pH G = 6.5, and
surprisingly, P H + decreased as
pH G decreased.
enhanced yellow fluorescent protein; ultraviolet enhanced green
fluorescent protein; trachea; organelle pH; proton permeability; cystic
fibrosis
The pH and trafficking of recycling endosomes have previously been studied using transferrin. We have used another approach, one in which the vesicle transport protein cellubrevin was appended with a ...luminal IgG epitope to allow targeting of fluorescein-5′-isothiocyanate (FITC)-labeled anti-IgG F(ab) antibodies to the recycling endosomes in living cells. FITC-F(ab) was specifically internalized by COS cells transfected with cellubrevin-Ig, which at steady state accumulated in a pericentriolar region similar to rhodamine-transferrin. Confocal microscopic analysis showed that endosome labeling by these two markers was heterogeneous. This differential distribution was not induced by the IgG tag, since endogenous Cb and Tf were also partitioned into separate endosomal populations. We used fluorescence ratio imaging of internalized FITC-F(ab) to measure the pH of cellubrevin-enriched recycling endosomes (pHCb) and FITC-transferrin to measure the pH of transferrin-enriched recycling endosomes (pHTf). In COS cells, cellubrevin endosomes (mean pHCb 6.1 ± 0.05; range, 5.2–6.6) were more acidic than transferrin endosomes (mean pHTf 6.5 ± 0.05; range, 5.6–7.2). Similar results were obtained in Chinese hamster ovary cells. Treatment with the vacuolar H+-ATPase inhibitor bafilomycin A1caused pHTf to increase (ΔpHTf = 1.2 pH units) to a greater extent than pHCb (ΔpHCb = 0.5 pH units). Furthermore, inhibition of the Na+/K+-ATPase by ouabain or acetylstrophanthidin caused pHTf to decrease by 0.6 pH units but had no effect on pHCb. Based on the combination of these morphological and functional data, we suggest that the recycling endosomes are heterogeneous in their biochemical compositions, ion transport properties, and pH values.
The Alliance for Cellular Signaling Plasmid Collection Zavzavadjian, Joelle R.; Couture, Sam; Park, Wei Sun ...
Molecular & cellular proteomics,
March 2007, 20070301, 2007-03-00, Letnik:
6, Številka:
3
Journal Article
Recenzirano
Odprti dostop
Cellular responses to inputs that vary both temporally and spatially are determined by complex relationships between the components of cell signaling networks. Analysis of these relationships ...requires access to a wide range of experimental reagents and techniques, including the ability to express the protein components of the model cells in a variety of contexts. As part of the Alliance for Cellular Signaling, we developed a robust method for cloning large numbers of signaling ORFs into Gateway® entry vectors, and we created a wide range of compatible expression platforms for proteomics applications. To date, we have generated over 3000 plasmids that are available to the scientific community via the American Type Culture Collection. We have established a website at www.signaling-gateway.org/data/plasmid/ that allows users to browse, search, and blast Alliance for Cellular Signaling plasmids. The collection primarily contains murine signaling ORFs with an emphasis on kinases and G protein signaling genes. Here we describe the cloning, databasing, and application of this proteomics resource for large scale subcellular localization screens in mammalian cell lines.
We report the isolation of a novel mouse voltage-gated Shaker-related K+ channel gene,Kv1.7 (Kcna7/KCNA7). Unlike other known Kv1 family genes that have intronless coding regions, the protein-coding ...region of Kv1.7 is interrupted by a 1.9-kilobase pair intron. The Kv1.7 gene and the related Kv3.3(Kcnc3/KCNC3) gene map to mouse chromosome 7 and human chromosome 19q13.3, a region that has been suggested to contain a diabetic susceptibility locus. The mouse Kv1.7 channel is voltage-dependent and rapidly inactivating, exhibits cumulative inactivation, and has a single channel conductance of 21 pS. It is potently blocked by noxiustoxin and stichodactylatoxin, and is insensitive to tetraethylammonium, kaliotoxin, and charybdotoxin. Northern blot analysis reveals ∼3-kilobase pair Kv1.7transcripts in mouse heart and skeletal muscle. In situhybridization demonstrates the presence of Kv1.7 in mouse pancreatic islet cells. Kv1.7 was also isolated from mouse brain and hamster insulinoma cells by polymerase chain reaction.
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