Warth, S. Adrenal aldosterone-producing adenomas APAs are a main cause for primary aldosteronism leading to arterial hypertension. Physiologically, aldosterone production in the adrenal gland is stimulated by angiotensin II and high extracellular potassium. In parallel to these findings, mRNA expression of the cytochrome P, family 11, subfamily B, polypeptide 2 aldosterone synthase was substantially increased and aldosterone production was enhanced in cells overexpressing mutant ATP2B3.
Therefore, the control of the membrane potential of glomerulosa cells is of upmost importance for the regulation of aldosterone production.
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In , the seminal work by Choi et al further emphasized this concept Since then, this finding has been corroborated in larger cohorts of patients 15 — In humans, ATP2B3 is expressed in all 3 zones of the adrenal cortex Two days before the experiment, 1. Transfection efficiency was verified by real-time PCR, Western blotting, and immunofluorescence staining Supplemental Figure 2. For life cell measurements, transfected cells were identified using anti-CD8-coated dynabeads Life Technologies GmbH or red fluorescence.
The animals had free access to food and water. The experimental protocols were approved by the local councils for animal care and were conducted according to the German laws for animal care and the NIH Guide for the Care and Use of Laboratory Animals. The preparation of adrenal cryosection specimen and immunofluorescence were performed as described previously Mean fluorescence ratios of emission at — nm after excitation at and nm were calculated for single transfected cells after subtraction of the background signal using the Axiovision software Zeiss.
Protein concentration of the cell lysate was measured using a standard Bradford assay Bio-Rad. The total amount of aldosterone measured in the medium was normalized against the total amount of cellular protein. Medium was collected for aldosterone measurements after a hour stimulation period. Aldosterone levels, normalized to total cell protein were calculated in relation to the aldosterone secretion measured in empty vector cells under control conditions.
Quality of the RNA used for reverse transcription was tested by agarose electrophoresis. Human adrenal tissue sampling was approved by the local ethics committee of the University Clinic Munich and all patients provided written informed consent. The specificity of PCR amplifications was verified by agarose electrophoresis and melting curve analysis. Primer sequences are listed in Supplemental Table 1. The following substances were used for the pharmacological characterization of mutated ATP2B3: 5-N-ethyl-N-Isopropyl-amiloride, flufenamic acid, lidocaine, triamterene, amiloride, benzamil, phenamil, bupivacain, diltiazem, nifedipine, verapamil, furosemide, and hydrochlorothiazide all from Sigma-Aldrich ; cariporide Santa Cruz Biotechnology, Inc ; 2-[2-[4- 4-nitrobenzyloxy phenyl]ethyl]isothiourea mesylate and 2-[[4-[ 4 nitrophenyl methoxy]phenyl]methyl]thiazolidinecarboxylic acid ethyl ester Tocris Bioscience.
Swelling and disruption of the cells was induced by incubation in deionized water on ice for 1.
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Lysates were homogenized with needle and syringe 27 G and cleared from cellular debris by centrifugation 10 min, 16 g. Separate sets of experiments were measured on different days using different passages of NCI-HR and HEK cells in order to prove reproducibility of the data.
Handbook of ATPases: Biochemistry, Cell Biology, Pathophysiology
Student's t test was used as appropriate to calculate the level of significance for single comparisons. Using immunofluorescence, the Atp2b3 protein was mainly localized in the plasma membrane of the aldosterone producing glomerulosa cells, which were marked by a Zg-specific Dab2 staining Figure 1 C. A weaker Atp2b3 signal was detectable in the outer cell layers of the mouse zona fasciculata. In the mouse, adrenal gland immunofluorescence for Atp2b3 red is restricted to the Zg and outer zona fasciculata C. The Zg cell marker Dab2 disabled homolog 2 44 is stained in green on consecutive sections.
The expression levels of other enzymes involved in aldosterone synthesis were not significantly changed Supplemental Table 3.
C, In a separate set of experiments, 24 hours after transfection the control medium 4. More recent studies have involved novel enzymes involved with cellulose degradation. David Ron is a Professor at Cambridge University. The lab uses biochemical, biophysical and cell-based tools to research both the molecular mechanisms that recognize the burden of unfolded proteins and thus initiate signalling in the ER unfolded protein response UPR and the downstream effector pathways by which cells adapt to unfolded protein stress in their ER.
These effector mechanisms engage post-translational regulation of ER chaperone function, regulated translation of mRNA and transcriptional control of gene expression and thus interface with other cellular stress pathways. To eLife , David Ron brings scientific expertise in the study of the unfolded protein response, chaperone function and stress-induced regulation of mRNA translation and editorial experience from having served as an eLife Reviewing Editor since Her earlier worked focused on the structural basis for combinatorial regulation of gene expression and the molecular mechanisms of the sirtuin family of protein deacetylases.
Her current research centers on the mechanisms by which ubiquitin plays a signaling role in transcription and the DNA damage response. All sections. He is a founder of Nurix San Francisco. He has served on the editorial board of Cell and was an associate editor of Molecular Biology of the Cell. Open in a separate window.
Figure 1. Three different topologies can be adopted by P-type ATPases in Mycobacterium tuberculosis The six different algorithms used in the hydrophobicity analysis showed that all of the M.
Figure 2. Figure 3. Figure 4. P-type ATPases of the Mycobacterium tuberculosis complex possess the characteristic motifs involved in cation transport The common catalytic mechanism of P-type ATPases is partially supported by conserved core sequences that are useful in the recognition of these pumps in proteomes [ 15 ]. Figure 5. Figure 6. Conclusion Mycobacteria are unicellular organisms that respond to environmental stimuli, and the transport of substances across the plasma membrane could play a fundamental role in their adaptability.
Competing interest There are no conflicts of interest to declare. References WHO. Global tuberculosis control: WHO report Switzerland: Publications of the World Health Organization; The role of IS in the evolution of Mycobacterium tuberculosis. Tuberculosis Edinb ; 87 5 — Detection and discrimination of Mycobacterium tuberculosis complex. Diagn Microbiol Infect Dis. Tuberculosis: pathophysiology, clinical features, and diagnosis. Crit Care Nurse. Latent tuberculosis: mechanisms of host and bacillus that contribute to persistent infection.
Lancet Infect Dis. The challenge of new drug discovery for tuberculosis. Comparative molecular biological analysis of membrane transport genes in organisms. Plant Mol Biol.
Handbook of ATPases : biochemistry, cell biology, pathophysiology
Evolution of substrate specificities in the P-type ATPase superfamily. J Mol Evol. Transport ATPases into the year a brief overview related to types, structures, functions and roles in health and disease. J Bioenerg Biomembr. Biochim Biophys Acta. J Exp Mar Bio Ecol. Conformational changes in the enzyme Transitions between the Na-form and the K-form studied with tryptic digestion as a tool. Annu Rev Physiol. J Mol Biol. P-type ATPases. Annu Rev Biophys. Biology, structure and mechanism of P-type ATPases. Nat Rev Mol Cell Biol.
In and out of the cation pumps: P-type ATPase structure revisited. Curr Opin Struct Biol. Profile hidden Markov models. BMC Bioinforma.
Antimony-Phosphomolybdate ATPase Assay
Bioinformatic characterization of P-type ATPases encoded within the fully sequenced genomes of 26 eukaryotes. J Membr Biol. The P-type ATPase superfamily. J Mol Microbiol Biotechnol. Deciphering the biology of Mycobacterium tuberculosis from the complete genome sequence. Mycobacterial p 1 -type ATPases mediate resistance to zinc poisoning in human macrophages. Cell Host Microbe.
A P-type ATPase importer that discriminates between essential and toxic transition metals. CtpV: a putative copper exporter required for full virulence of Mycobacterium tuberculosis. Mol Microbiol. Proc Natl Acad Sci.