The publication data currently available has been vetted by Vanderbilt faculty, staff, administrators and trainees. The data itself is retrieved directly from NCBI's PubMed and is automatically updated on a weekly basis to ensure accuracy and completeness.
If you have any questions or comments, please contact us.
We recently reported the case of a young patient with multisystem failure carrying a de novo mutation in SLC12A2, the gene encoding the Na-K-2Cl cotransporter-1 (NKCC1). Heterologous expression studies in nonepithelial cells failed to demonstrate dominant-negative effects. In this study, we examined expression of the mutant cotransporter in epithelial cells. Using Madin-Darby canine kidney (MDCK) cells grown on glass coverslips, permeabilized support, and Matrigel, we show that the fluorescently tagged mutant cotransporter is expressed in cytoplasm and at the apical membrane and affects epithelium integrity. Expression of the mutant transporter at the apical membrane also results in the mislocalization of some of the wild-type transporter to the apical membrane. This mistargeting is specific to NKCC1 as the Na-K-ATPase remains localized on the basolateral membrane. To assess transporter localization in vivo, we created a mouse model using CRISPR/cas9 that reproduces the 11 bp deletion in exon 22 of Slc12a2. Although the mice do not display an overt phenotype, we show that the colon and salivary gland expresses wild-type NKCC1 abundantly at the apical pole, confirming the data obtained in cultured epithelial cells. Enough cotransporter must remain, however, on the basolateral membrane to participate in saliva secretion, as no significant decrease in saliva production was observed in the mutant mice.
Loss-of-function mutations in the potassium-chloride cotransporter KCC3 lead to Andermann syndrome, a severe sensorimotor neuropathy characterized by areflexia, amyotrophy and locomotor abnormalities. The molecular events responsible for axonal loss remain poorly understood. Here, we establish that global or neuron-specific KCC3 loss-of-function in mice leads to early neuromuscular junction (NMJ) abnormalities and muscular atrophy that are consistent with the pre-synaptic neurotransmission defects observed in patients. KCC3 depletion does not modify chloride handling, but promotes an abnormal electrical activity among primary motoneurons and mislocalization of Na/K-ATPase α1 in spinal cord motoneurons. Moreover, the activity-targeting drug carbamazepine restores Na/K-ATPase α1 localization and reduces NMJ denervation in Slc12a6 mice. We here propose that abnormal motoneuron electrical activity contributes to the peripheral neuropathy observed in Andermann syndrome.
Copyright © 2017 Elsevier Inc. All rights reserved.
Acute Helicobacter pylori infection of gastric epithelial cells and human gastric biopsies represses H,K-ATPase α subunit (HKα) gene expression and inhibits acid secretion, causing transient hypochlorhydria and supporting gastric H. pylori colonization. Infection by H. pylori strains deficient in the cag pathogenicity island (cag PAI) genes cagL, cagE, or cagM, which do not transfer CagA into host cells or induce interleukin-8 secretion, does not inhibit HKα expression, nor does a cagA-deficient strain that induces IL-8. To test the hypothesis that virulence factors other than those mediating CagA translocation or IL-8 induction participate in HKα repression by activating NF-κB, AGS cells transfected with HKα promoter-Luc reporter constructs containing an intact or mutated NF-κB binding site were infected with wild-type H. pylori strain 7.13, isogenic mutants lacking cag PAI genes responsible for CagA translocation and/or IL-8 induction (cagA, cagζ, cagε, cagZ, and cagβ), or deficient in genes encoding two peptidoglycan hydrolases (slt and cagγ). H. pylori-induced AGS cell HKα promoter activities, translocated CagA, and IL-8 secretion were measured by luminometry, immunoblotting, and ELISA, respectively. Human gastric biopsy acid secretion was measured by microphysiometry. Taken together, the data showed that HKα repression is independent of IL-8 expression, and that CagA translocation together with H. pylori transglycosylases encoded by slt and cagγ participate in NF-κB-dependent HKα repression and acid inhibition. The findings are significant because H. pylori factors other than CagA and IL-8 secretion are now implicated in transient hypochlorhydria which facilitates gastric colonization and potential triggering of epithelial progression to neoplasia.
Copyright © 2015 the American Physiological Society.
INTRODUCTION - To investigate tissue flow disturbance and hypoxia during migraine aura, we studied a case of familial hemiplegic migraine (FHM) using novel magnetic resonance imaging (MRI) techniques.
CASE RESULTS - A 44-year-old male was admitted with suspected stroke because of confusion and aphasia. Initial gadolinium-based perfusion MRI showed a decrease in cerebral blood flow and an increase in capillary flow disturbances within the left hemisphere. Later during the prolonged aura phase, chemical exchange saturation transfer MRI indicated a drop in pH in the affected area. The patient was diagnosed with an R908Q mutation in the ATP1A2 gene causing FHM type 2.
DISCUSSION - During prolonged aura in FHM, MRI shows reduced CBF, capillary flow disturbances and a possible pH drop that could indicate tissue hypoxia.
© International Headache Society 2015.
AIMS/HYPOTHESIS - Non-invasive imaging of the pancreatic beta cell mass (BCM) requires the identification of novel and specific beta cell biomarkers. We have developed a systems biology approach to the identification of promising beta cell markers.
METHODS - We followed a functional genomics strategy based on massive parallel signal sequencing (MPSS) and microarray data obtained in human islets, purified primary rat beta cells, non-beta cells and INS-1E cells to identify promising beta cell markers. Candidate biomarkers were validated and screened using established human and macaque (Macacus cynomolgus) tissue microarrays.
RESULTS - After a series of filtering steps, 12 beta cell-specific membrane proteins were identified. For four of the proteins we selected or produced antibodies targeting specifically the human proteins and their splice variants; all four candidates were confirmed as islet-specific in human pancreas. Two splice variants of FXYD domain containing ion transport regulator 2 (FXYD2), a regulating subunit of the Na(+)-K(+)-ATPase, were identified as preferentially present in human pancreatic islets. The presence of FXYD2gammaa was restricted to pancreatic islets and selectively detected in pancreatic beta cells. Analysis of human fetal pancreas samples showed the presence of FXYD2gammaa at an early stage (15 weeks). Histological examination of pancreatic sections from individuals with type 1 diabetes or sections from pancreases of streptozotocin-treated Macacus cynomolgus monkeys indicated a close correlation between loss of FXYD2gammaa and loss of insulin-positive cells.
CONCLUSIONS/INTERPRETATION - We propose human FXYD2gammaa as a novel beta cell-specific biomarker.
Muscle contraction and metabolic stress are potent activators of AMP-activated protein kinase (AMPK). AMPK restores energy balance by activating processes that produce energy while inhibiting those that consume energy. The role of AMPK in the regulation of active ion transport is unclear. Our aim was to determine the effect of the AMPK activator A-769662 on Na(+)-K(+)-ATPase function in skeletal muscle cells. Short-term incubation of differentiated rat L6 myotubes with 100 microM A-769662 increased AMPK and acetyl-CoA carboxylase (ACC) phosphorylation in parallel with decreased Na(+)-K(+)-ATPase alpha(1)-subunit abundance at the plasma membrane and ouabain-sensitive (86)Rb(+) uptake. Notably, the effect of A-769662 on Na(+)-K(+)-ATPase was similar in muscle cells that do not express AMPK alpha(1)- and alpha(2)-catalytic subunits. A-769662 directly inhibits the alpha(1)-isoform of the Na(+)-K(+)-ATPase, purified from rat and human kidney cells in vitro with IC(50) 57 microM and 220 microM, respectively. Inhibition of the Na(+)-K(+)-ATPase by 100 microM ouabain decreases sodium pump activity and cell surface abundance, similar to the effect of A-769662, without affecting AMPK and ACC phosphorylation. In conclusion, the AMPK activator A-769662 inhibits Na(+)-K(+)-ATPase activity and decreases the sodium pump cell surface abundance in L6 skeletal muscle cells. The effect of A-769662 on sodium pump is due to direct inhibition of the Na(+)-K(+)-ATPase activity, rather than AMPK activation. This AMPK-independent effect on Na(+)-K(+)-ATPase calls into question the use of A-769662 as a specific AMPK activator for metabolic studies.
Aberrant N-linked glycans promote the malignant potential of cells by enhancing the epithelial-to-mesenchymal transition and the invasive phenotype. To identify small molecule inhibitors of N-glycan biosynthesis, we developed a chemical screen based on the ability of the tetravalent plant lectin L-phytohemagglutinin (L-PHA) to bind and crosslink surface glycoproteins with beta1,6GlcNAc-branched complex type N-glycans and thereby induce agglutination and cell death. In this screen, Jurkat cells were treated with a library of off-patent chemicals (n = 1,280) to identify molecules that blocked L-PHA-induced death. The most potent hit from this screen was the cardiac glycoside (CG) dihydroouabain. In secondary assays, a panel of CGs was tested for their effects on L-PHA-induced agglutination and cell death. All of the CGs tested inhibited L-PHA-induced death in Jurkat cells, and the most potent CG tested was digoxin with an EC(50) of 60 +/- 20 nmol/L. Digoxin also increased the fraction of some concanavalin A-binding N-glycans. Using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, digoxin specifically increased GlcNAc(1)Man(3)GlcNAc(2)Fuc(1) and GlcNAc(2)Man(3)GlcNAc(2)Fuc(1) oligosaccharides demonstrating an impairment of the N-glycan pathway. Consistent with this effect on the N-glycan pathway, digoxin inhibited N-glycosylation-mediated processes of tumor cell migration and invasion. Furthermore, digoxin prevented distant tumor formation in two mouse models of metastatic prostate cancer. Thus, taken together, our high throughput screen identified CGs as modifiers of the N-glycan pathway. These molecules can be used as tools to better understand the role of N-glycans in normal and malignant cells. Moreover, these results may partly explain the anticancer effect of CGs in cardiovascular patients.
p120 catenin is thought to be a key regulator of E-cadherin function and stability, but its role(s) in vivo is poorly understood. To examine these directly, we generated a conditional p120 knockout mouse and targeted p120 ablation to the embryonic salivary gland. Surprisingly, acinar differentiation is completely blocked, resulting in a gland composed entirely of ducts. Moreover, p120 ablation causes E-cadherin deficiency in vivo and severe defects in adhesion, cell polarity, and epithelial morphology. These changes closely phenocopy high-grade intraepithelial neoplasia, a condition that, in humans, typically progresses to invasive cancer. Tumor-like protrusions appear immediately after p120 ablation at e14 and expand into the lumen until shortly after birth, at which time the animals die with completely occluded glands. The data reveal an unexpected role for p120 in salivary acinar development and show that p120 ablation by itself induces effects consistent with a role in tumor progression.
In Cerro de Pasco (CP), Peru (altitude 4338 m) 24% of men have migraine with aura. We studied 30 men. Twenty CP natives, examined in CP, were rated using a chronic mountain sickness (CMS) score to separate controls (10) from those with CMS (10), a maladaptation syndrome in natives to altitude which includes severe, recurring headache. We collected white cells in CP and, from the same men, within 1 h of arrival in Lima (150 m above sea level). Ten normal US men volunteered white cells for comparison. After RNA extraction we assessed gene expression by reverse transcription-polymerase chain reaction. Low ATP1A1 subunit of the ATPase gene mRNA expression in CP was correlated with headache (P=0.002), acral paraesthesias (P=0.004) and CMS score (P<0.001). ATP1A1 subunit expression was increased in all Andeans in Lima (P<0.001). There were no differences between Andean controls in Lima and US controls. Manipulation of Na+/K+ATPase could offer relief for migraineurs at sea level.
BACKGROUND - Thirty-nine percent of permanent altitude dwellers in the Andes experience acral paresthesias.
METHODS - Clinical examinations, sural nerve biopsies, and electrodiagnostic studies on peripheral nerves were performed on 15 men. Ten Cerro de Pasco (CP) natives living at 4,338 meters were biopsied. Three of these subjects had no burning feet/burning hands (BF/BH); three had BF/BH; and four had chronic mountain sickness (CMS), a maladaptation syndrome resulting from living in the Andes, all with BF/BH. Three patients with CMS were biopsied in Lima within hours after leaving CP. Two normal Lima natives were biopsied in Lima. Symptom scores for BF/BH and CMS score ratings were used. The nerves were assayed for Na+, K+ adenosine triphosphatase (ATPase), cytochrome oxidase (CO), substance P (SP), and endothelin (ET).
RESULTS - Low ATPase was inversely related to symptom scores and CMS scores (p < 0.001). Patients with CMS biopsied in normoxia (Lima) had ATPase levels similar to those of controls. Nerve motor conduction velocities and sensory action potentials were normal. CO was inversely related to age (p < 0.03) and no relation of SP to any variable was found. ET levels were lower in sea level natives (p = 0.04).
CONCLUSIONS - Acral paresthesias are associated with low ATPase in peripheral nerves. Lower ET levels of sea level natives likely reflect lowered release from vasa nervorum.