Key points
Gain‐of‐function mutations in the highly selective Ca2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping.
TAM‐associated mutations ...in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca2+ influx and increasing the currents evoked by Ca2+ store depletion.
Mutation V107M additionally decreases the channel selectivity for Ca2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species.
The ORAI blocker GSK‐7975A prevents the constitutive activity of TAM‐associated channels and might be used in therapy for patients suffering from TAM.
Skeletal muscle differentiation relies on store‐operated Ca2+ entry (SOCE) mediated by STIM proteins linking the depletion of endoplasmic/sarcoplasmic reticulum Ca2+ stores to the activation of membrane Ca2+‐permeable ORAI channels. Gain‐of‐function mutations in STIM1 or ORAI1 isoforms cause tubular aggregate myopathy (TAM), a skeletal muscle disorder with muscular pain, weakness and cramping. Here, we characterize two overactive ORAI1 mutants from patients with TAM: V107M and T184M, located in the first and third transmembrane domain of the channel. When ectopically expressed in HEK‐293T cells or human primary myoblasts, the mutated channels increased basal and store‐operated Ca2+ entry. The constitutive activity of V107M, L138F, T184M and P245L mutants was prevented by low concentrations of GSK‐7975A while the G98S mutant was resistant to inhibition. Electrophysiological recordings confirmed ORAI1‐V107M constitutive activity and revealed larger STIM1‐gated V107M‐ and T184M‐mediated currents with conserved fast and slow Ca2+‐dependent inactivation. Mutation V107M altered the channel selectivity for Ca2+ ions and conferred resistance to acidic inhibition. Ca2+ imaging and molecular dynamics simulations showed a preserved sensitivity of T184M to the negative regulation by reactive oxygen species. Both mutants were able to mediate SOCE in Stim1−/−/Stim2−/− mouse embryonic fibroblasts expressing the binding‐deficient STIM1‐F394H mutant, indicating a higher sensitivity for STIM1‐mediated gating, with ORAI1‐T184M gain‐of‐function being strictly dependent on STIM1. These findings provide new insights into the permeation and regulatory properties of ORAI1 mutants that might translate into therapies against diseases with gain‐of‐function mutations in ORAI1.
Key points
Gain‐of‐function mutations in the highly selective Ca2+ channel ORAI1 cause tubular aggregate myopathy (TAM) characterized by muscular pain, weakness and cramping.
TAM‐associated mutations in ORAI1 first and third transmembrane domain facilitate channel opening by STIM1, causing constitutive Ca2+ influx and increasing the currents evoked by Ca2+ store depletion.
Mutation V107M additionally decreases the channel selectivity for Ca2+ ions and its inhibition by acidic pH, while mutation T184M does not alter the channel sensitivity to pH or to reactive oxygen species.
The ORAI blocker GSK‐7975A prevents the constitutive activity of TAM‐associated channels and might be used in therapy for patients suffering from TAM.
Background: Vascular endothelial growth factor (VEGF) and histamine induce von Willebrand factor (VWF) release from vascular endothelial cells. Protein kinase C (PKC) is involved in the control of ...exocytosis in many secretory cell types. Objectives: We investigated the role of PKC and the interactions between PKC and Ca2+ signaling in both VEGF‐induced and histamine‐induced VWF secretion from human umbilical vein endothelial cells (HUVECs). Results: Several PKC inhibitors (staurosporine, Ro31‐8220, myristoylated PKC peptide inhibitor and Go6983) block VEGF‐induced but not histamine‐induced VWF secretion. PKC‐α and novel PKCs (PKC‐δ, PKC‐ε, and PKC‐η), but not PKC‐β, are expressed in HUVECs. Both VEGF and histamine activate PKC‐δ. However, gene inactivation experiments using small interfering RNA indicate that PKC‐δ (but not PKC‐α) is involved in the regulation of VEGF‐induced but not histamine‐induced secretion. Both VEGF and histamine induce a rise in cytosolic free Ca2+ (Ca2+c), but the response to VEGF is weaker and even absent in a significant subset of cells. Furthermore, VEGF‐induced secretion is largely preserved when the rise in Ca2+c is prevented by BAPTA‐AM. Conclusions: Our study identifies striking agonist specificities in signal–secretion coupling. Histamine‐induced secretion is dependent on Ca2+c but not PKC, whereas VEGF‐induced secretion is largely dependent on PKC‐δ and significantly less on Ca2+c. Our data firmly establish the key role of PKC‐δ in VEGF‐induced VWF release, but suggest that a third, VEGF‐specific, signaling intermediate is required as a PKC‐δ coactivator.
Objective To investigate the interactions between the P. aeruginosa N-3-oxo-dodecanoyl-L-homoserine lactone (C12) quorum-sensing molecule and human airway epithelial cell gap junctional intercellular ...communication (GJIC). Methods C12 degradation and its effects on cells were monitored in various human airway epithelial cell models grown under non-polarized and polarized conditions. Its concentration was further monitored in daily tracheal aspirates of colonized intubated patients. Results C12 rapidly altered epithelial integrity and decreased GJIC in non-polarized cells while other quorum-sensing molecules had no effect. C12 increased Ca2+ i by promoting calcium release from the ER and calcium influx. C12 cytotoxic effects were dependent on Ca2+ i and could be prevented by inhibitors of Src and Rho-associated protein kinases. In contrast, polarized airway cells grown on Transwell filters were protected from C12. In vivo during colonization of intubated patients, C12 did not accumulate, but paralleled bacterial densities. Interestingly, in vitro C12 degradation, a reaction catalyzed by intracellular paraoxonase 2 (PON2), was impaired in non-polarized cells whereas PON2 expression was increased during epithelial polarization. Finally, in a wound injury model of primary airway epithelial cells grown at the air-liquid interface, repeated exposure of C12 impairs airway epithelial cell repair. Conclusion The cytotoxicity of C12 on non-polarized epithelial cells, combined with its impaired degradation allowing its accumulation, provide an additional pathogenic mechanism for P. aeruginosa infections.
Oligonucleotides containing Locked Nucleic Acids (LNA) to various extents and at various positions were evaluated for antisense activity, RNase H recruitment, nuclease stability and thermal affinity. ...In this work, two different diastereoisomers of LNA were studied: the beta‐d‐LNA and the alpha‐l‐LNA (abbreviated as β‐d‐LNA and α‐l‐LNA). Our findings show that the best antisense activity with 16mer gapmers containing β‐d‐LNA (oligonucleotides containing consecutive segments of LNA and DNA with a central DNA stretch flanked by two LNA segments, LNA–DNA–LNA) is found with gap sizes between 7 and 10 nt. The optimal gap size is motif‐dependent, and requires the right balance between gap size and affinity. Compared to β‐d‐LNA, α‐l‐LNA shows superior stability against a 3′‐exonuclease. The design possibilities of α‐l‐LNA were explored for different gapmers and other designs, collectively called chimeras. The placement of α‐l‐LNA in the junctions or in the flanks resulted in potent antisense oligonucleotides. Moreover, different chimeras with an alternate composition of DNA, α‐l‐LNA and β‐d‐LNA were evaluated in terms of antisense activity and RNase H recruitment. Chimeras with an interrupted DNA stretch with α‐l‐LNA still recruit RNase H and show good levels of antisense activity, while the same design with β‐d‐LNA results in a drop in antisense potency. Our findings indicate that α‐l‐LNA is a powerful and versatile nucleotide analogue for designing potent antisense oligonucleotides.
Objective: von Willebrand factor (VWF) is acutely released from endothelial cells in response to numerous calcium‐raising agents (e.g. thrombin, histamine) and cAMP‐raising agents (e.g. epinephrine, ...adenosine, vasopressin). In contrast, very few inhibitors of endothelial VWF secretion have been described. The neurotransmitter dopamine is a modulator of exocytosis in several endocrine cells, and is possibly involved in the regulation of several endothelial cell functions. We therefore investigated the effect of dopamine on endothelial VWF secretion. Results: Dopamine, D2/D3‐ and D4‐specific agonists inhibited histamine‐ but not thrombin‐induced VWF secretion. Expression of dopamine D2, D3 and D4 receptors was demonstrated by reverse transcription polymerase chain reaction (RT‐PCR) in both human aortic (HAEC) and umbilical vein (HUVEC) endothelial cells. D2–D4 agonists did not inhibit histamine‐induced rise in Ca2+i: they inhibited histamine‐induced secretion even in the absence of extracellular calcium. Thus, the dopamine effects are not mediated by Ca2+i‐dependent signalling. D2/D3‐ and D4‐specific agonists inhibited neither the rise in cAMP nor VWF secretion in response to epinephrine and adenosine, arguing against an effect on cAMP‐mediated signalling. D1 and D5 receptors were not detected in HAEC or HUVEC by RT‐PCR, and the D1/D5‐specific agonist SKF 38 393 failed to modulate VWF secretion, arguing against a role for these receptors in endothelial exocytosis. Conclusions: Dopamine inhibits histamine‐induced endothelial exocytosis by activating D2–D4 receptor, via a mechanism distinct from Ca2+i‐or cAMP‐mediated signaling. In contrast, D1 and D5 receptors are not functionally expressed in cultured endothelial cells. Dopamine agonists may be useful as inhibitors of endothelial activation in inflammation and cardiovascular disease.
Financial distress is a primary concern for young adults with cancer.
The aim of this study was to identify material resources, physical and psychological health, and workplace variables that are ...associated with financial distress in young adult cancer survivors.
A cross-sectional study was conducted using the Cancer Survivor Employment Needs Survey. Participants were young adults (18-39 years of age) who lived in the United States and had a cancer diagnosis. Multivariable linear regression was used to model relations between financial distress and material resources, physical and psychological health, and workplace variables.
Participants (N = 214) were mostly non-Hispanic White (78%), female (79%), and had a mean age of 31 years and 4.6 years post-diagnosis. Material resources, physical and psychological health, and workplace variables were all identified as contributing to study participants' financial distress. Among the young adults surveyed, financial distress was prevalent, and an array of problems were associated with financial distress.
Oncology and rehabilitation providers should openly discuss finances with YAs with cancer and guide them to resources that can address their financial, benefits, and vocational needs to ultimately improve quality of life.
Abstract In this study the relationship between the efficiency of endoplasmic reticulum (ER) Ca2+ refilling and the extent of Ca2+ entry was investigated in endothelial cells. ER and mitochondrial ...Ca2+ concentration were measured using genetically encoded Ca2+ sensors, while the amount of entering Ca2+ was controlled by varying either the extracellular Ca2+ or the electrical driving force for Ca2+ by changing the plasma membrane potential. In the absence of an agonist, ER Ca2+ replenishment was fully accomplished even if the Ca2+ concentration applied was reduced from 2 to 0.5 mM. A similar strong efficiency of ER Ca2+ refilling was obtained under condition of plasma membrane depolarization. However, in the presence of histamine, ER Ca2+ refilling depended on mitochondrial Ca2+ transport and was more susceptible to membrane depolarization. Store-operated Ca2+ entry (SOCE), was strongly reduced under low Ca2+ and depolarizing conditions but increased if ER Ca2+ uptake was blocked or if ER Ca2+ was released continuously by IP3 . A correlation of the kinetics of ER Ca2+ refilling with cytosolic Ca2+ signals revealed that termination of SOCE is a rapid event that is not delayed compared to ER refilling. Our data indicate that ER refilling occurs in priority to, and independently from the cytosolic Ca2+ elevation upon Ca2+ entry and that this important process is widely achieved even under conditions of diminished Ca2+ entry.
This history of the medical profession in pre-Revolutionary Russia examines an influential segment of the educated elite. The author shows how Russian physicians differed in social origin, careers, ...and professionalization from their counterparts in other lands.
Originally published in 1982.
ThePrinceton Legacy Libraryuses the latest print-on-demand technology to again make available previously out-of-print books from the distinguished backlist of Princeton University Press. These paperback editions preserve the original texts of these important books while presenting them in durable paperback editions. The goal of the Princeton Legacy Library is to vastly increase access to the rich scholarly heritage found in the thousands of books published by Princeton University Press since its founding in 1905.
1. Epoxyeicosatrienoic acids (EETs) have been described as endothelium-derived hyperpolarizing factors (EDHFs), based on their
stimulatory effects on smooth muscle K+ channels. In order to reveal a ...putative autocrine effect of EETs on endothelial channels,
we have studied the effects of the four EET regioisomers (5,6-EET, 8,9-EET, 11,12-EET and 14,15-EET) on the high-conductance,
Ca(2+)-dependent K+ (BKCa) channel recorded in inside-out patches of primary cultured pig coronary artery endothelial cells.
Currents were recorded in the presence of either 500 nm or 1 microM free Ca2+ on the cytosolic side of the membrane. 2. In
81% of experiments, EETs at < 156 nM, applied on the cytosolic side of the membrane, transiently increased BKCa channel open
state probability (PO) without affecting its unitary conductance, thus providing evidence for direct action of EETs, without
involvement of a cytosolic transduction pathway. 3. The four EET regioisomers appeared to be equally active, multiplying the
BKCa channel PO by a mean factor of 4.3 +/- 0.6 (n = 15), and involving an increase in the number and duration of openings.
4. The EET-induced increase in BKCa channel activity was more pronounced with low initial PO. When the BKCa channel was activated
by 500 nM Ca2+, application of EETs increased the initial PO value of below 0.1 by a factor of 5. When the channel was activated
by 1 microM Ca2+, application of EETs increased the initial PO value by a factor of 3. 5. Our results show that EETs potentiate
endothelial BKCa channel activation by Ca2+. The autocrine action of EETs on endothelial cells, which occurs in the same concentration
range as their action on muscle cells, should therefore fully participate in the vasoactive effects of EETs, and thus be taken
into account when considering their putative EDHF function.
1
Epoxyeicosatrienoic acids (EETs) have been described as endothelium‐derived hyper‐polarizing factors (EDHFs), based on their stimulatory effects on smooth muscle K+channels. In order to reveal a ...putative autocrine effect of EETs on endothelial channels, we have studied the effects of the four EET regioisomers (5,6‐EET, 8,9‐EET, 11,12‐EET and 14,15‐EET) on the high‐conductance, Ca2+‐dependent K+ (BKCa) channel recorded in inside‐out patches of primary cultured pig coronary artery endothelial cells. Currents were recorded in the presence of either 500 nm or 1 μm free Ca2+ on the cytosolic side of the membrane.
2
In 81% of experiments, EETs at < 156 nm, applied on the cytosolic side of the membrane, transiently increased BKCa channel open state probability (P0) without affecting its unitary conductance, thus providing evidence for direct action of EETs, without involvement of a cytosolic transduction pathway.
3
The four EET regioisomers appeared to be equally active, multiplying the BKCa channel Po by a mean factor of 4.3 ± 0.6 (n= 15), and involving an increase in the number and duration of openings.
4
The EET‐induced increase in BKCa channel activity was more pronounced with low initial Po. When the BKCa channel was activated by 500 nm Ca2+, application of EETs increased the initial Po value of below 0.1 by a factor of 5. When the channel was activated by 1 μm Ca2+, application of EETs increased the initial Po value by a factor of 3.
5
Our results show that EETs potentiate endothelial BKCa channel activation by Ca2+. The autocrine action of EETs on endothelial cells, which occurs in the same concentration range as their action on muscle cells, should therefore fully participate in the vasoactive effects of EETs, and thus be taken into account when considering their putative EDHF function.
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