Inside-out Mode Research Articles

Influence of utilizing hemodialysis membranes outside-in on solute clearance and filtration efficiency – One step towards a novel combined lung and kidney support device

Many membrane oxygenator patients suffer from renal disfunction. For these patients, a novel device integrating artificial lung and kidney support is being developed. Although outside-in blood flow is standard for membrane oxygenators, this is not typical for hemodialysis systems. The effect of outside-in blood flow on the efficiency of hemodialysis fibers for continuous hemodialysis and hemofiltration is yet unclear.This study evaluates the efficiency of commercial hemodialyzer membranes utilized outside-in compared to traditional inside-out mode regarding clearance of urea and creatinine, and ultrafiltration coefficient during in-vitro tests with porcine blood. Our results showed that dialyzers (1.2 m2, asymmetric hollow fibers) utilized outside-in had similar clearances of urea and creatinine compared to dialyzers used in the traditional mode (p > 0.7). However, outside-in dialyzers had an ultrafiltration coefficient four times lower than dialyzers applied in a conventional way, but adequate fluid removal could be achieved by controlling pressures in the system. This in-vitro study indicates that outside-in fibers could be sufficiently effective to maintain typical continuous renal replacement therapy doses. We regard this as one step towards a novel device with a mixed membrane fiber bundle utilizing blood flow outside both hemodialysis fibers and gas exchange fibers to provide simultaneous lung and kidney support.

Comparative proteomics of vesicles essential for the egress of Plasmodium falciparum gametocytes from red blood cells.

Transmission of malaria parasites to the mosquito is mediated by sexual precursor cells, the gametocytes. Upon entering the mosquito midgut, the gametocytes egress from the enveloping erythrocyte while passing through gametogenesis. Egress follows an inside-out mode during which the membrane of the parasitophorous vacuole (PV) ruptures prior to the erythrocyte membrane. Membrane rupture requires exocytosis of specialized egress vesicles of the parasites; that is, osmiophilic bodies (OBs) involved in rupturing the PV membrane, and vesicles that harbor the perforin-like protein PPLP2 (here termed P-EVs) required for erythrocyte lysis. While some OB proteins have been identified, like G377 and MDV1/Peg3, the majority of egress vesicle-resident proteins is yet unknown. Here, we used high-resolution imaging and BioID methods to study the two egress vesicle types in Plasmodium falciparum gametocytes. We show that OB exocytosis precedes discharge of the P-EVs and that exocytosis of the P-EVs, but not of the OBs, is calcium sensitive. Both vesicle types exhibit distinct proteomes with the majority of proteins located in the OBs. In addition to known egress-related proteins, we identified novel components of OBs and P-EVs, including vesicle-trafficking proteins. Our data provide insight into the immense molecular machinery required for the inside-out egress of P. falciparum gametocytes.

TRP434 and TRP435 residues are crucial for sensing calcium ions and PI(4,5)P2 molecules in TRPC5 channel.

Transient receptor potential canonical (TRPC) channels are non-selective calcium-permeable cation channels. Among 7 subunits, TRPC4, 5 channels are well known to be potentiated by direct bindings of calcium ion and phosphatidylinositol 4,5-biphosphate. Both calcium and PIP2 were therefore also implicated in activation and desensitization of the channel. The binding site for calcium ion was revealed but PIP2 binding site remains controversial. Previously, we found two tryptophan residues in TRPC4 channel as a candidate by which calcium sensing in S2-S3 linker is delivered to channel gating. Here, we suggest the WW site in TRPC5 channel conducts delivering of calcium sensing like in TRPC4, and also acts as a potential PIP2 binding site. We made two single mutants (W434A, W435A) and a double mutant (WW/AA) from human TRPC5 channel. Wild-type and mutant TRPC5 channels were expressed in HEK293 cells for macroscopic current recordings in whole-cell or inside-out mode. Generally, our data demonstrate W434 and W435 residues participate in PIP2 binding and calcium sensing. Interestingly, when co-expressed with human muscarinic acetylcholine receptor type 3 (mAChR3) and stimulated by carbachol, W434A channel didn’t show desensitization characteristically observed in wild-type TRPC5 channel. Loss of desensitization in W434A channel implies that it has lost the ability of sensing PIP2 hydrolysis by PLC or the ability of phosphorylation by PKC. The latter possibility is ruled out because the C-terminus of the channels we used were truncated so they do not contain the phosphorylation site. Recently, PIP2-bound ion channel structures were revealed including TRPs. Many channels had PIP2 molecules near S3, S4, and S5 helices. Our electrophysiological data also indicate that PIP2 binds near S3 helix in TRPC5 channel, and the WW site is a specific candidate for sensing PIP2 and calcium.

Vesicle dynamics during the egress of malaria gametocytes from the red blood cell

Malaria parasites are obligate intracellular pathogens that live in human red blood cells harbored by a parasitophorous vacuole. The parasites need to exit from the red blood cell to continue life-cycle progression and ensure human-to-mosquito transmission. Two types of blood stages are able to lyse the enveloping red blood cell to mediate egress, the merozoites and the gametocytes. The intraerythrocytic parasites exit the red blood cell via an inside-out mode during which the membrane of the parasitophorous vacuole ruptures prior to the red blood cell membrane. Membrane rupture is initiated by the exocytosis of specialized secretory vesicles following the perception of egress triggers. The molecular mechanisms of red blood cell egress have particularly been studied in malaria gametocytes. Upon activation by external factors, gametocytes successively discharge at least two types of vesicles, the osmiophilic bodies needed to rupture the parasitophorous vacuole membrane and recently identified egress vesicles that are important for the perforation of the erythrocyte membrane. In recent years, important components of the signaling cascades leading to red blood cell egress have been investigated and several proteins of the osmiophilic bodies have been identified. We here report on the newest findings on the egress of gametocytes from the red blood cell. We further focus on the content and function of the egress-related vesicles and discuss the molecular machinery that might drive vesicle discharge.

Effective Activation of BKCa Channels by QO-40 (5-(Chloromethyl)-3-(Naphthalen-1-yl)-2-(Trifluoromethyl)Pyrazolo [1,5-a]pyrimidin-7(4H)-one), Known to Be an Opener of KCNQ2/Q3 Channels.

QO-40 (5-(chloromethyl)-3-(naphthalene-1-yl)-2-(trifluoromethyl) pyrazolo[1,5-a]pyrimidin-7(4H)-one) is a novel and selective activator of KCNQ2/KCNQ3 K+ channels. However, it remains largely unknown whether this compound can modify any other type of plasmalemmal ionic channel. The effects of QO-40 on ion channels in pituitary GH3 lactotrophs were investigated in this study. QO-40 stimulated Ca2+-activated K+ current (IK(Ca)) with an EC50 value of 2.3 μM in these cells. QO-40-stimulated IK(Ca) was attenuated by the further addition of GAL-021 or paxilline but not by linopirdine or TRAM-34. In inside-out mode, this compound added to the intracellular leaflet of the detached patches stimulated large-conductance Ca2+-activated K+ (BKCa) channels with no change in single-channel conductance; however, there was a decrease in the slow component of the mean closed time of BKCa channels. The KD value required for the QO-40-mediated decrease in the slow component at the mean closure time was 1.96 μM. This compound shifted the steady-state activation curve of BKCa channels to a less positive voltage and decreased the gating charge of the channel. The application of QO-40 also increased the hysteretic strength of BKCa channels elicited by a long-lasting isosceles-triangular ramp voltage. In HEK293T cells expressing α-hSlo, QO-40 stimulated BKCa channel activity. Overall, these findings demonstrate that QO-40 can interact directly with the BKCa channel to increase the amplitude of IK(Ca) in GH3 cells.

Physiological evidence of mitochondrial permeability transition pore opening caused by lipid deposition leading to hepatic steatosis in db/db mice

Mitochondrial permeability transition pore (mPTP) is an important regulator in cell apoptosis and necrosis. However, its role in hepatic steatosis, especially its electrophysiological properties transformation remains elusive. Herein, using diabetes mice, we investigated the role of mPTP in hepatic steatosis triggered by diabetes and the mechanisms involved. We found that hepatic steatosis altered mitochondrial morphology, generating mega mitochondria, mitochondria swelling, calcein fluorescence quenching and mitochondrial membrane potential depolarization. At the same time, we confirmed an augmented mPTP opening with patch clamping in liver mitoplasts in db/db mice and a similar transformation with arachidonic acid (AA) simulating liquid deposition. We also found mPTP opening was significantly attenuated in wt mice after removing mitochondrial matrix, while that in db/db mice remained active. In addition, we observed that AA could directly activate mPTP in inside-out mode, independent of matrix calcium. In conclusion, we for the first time provided a physiological evidence of mPTP opening in lipid deposition, which could be directly induced by AA without Ca2+ and can be inhibited by cyclosporine A. As a result, it led to mitochondria morphology and function transformation. This might provide insights into potential therapeutic target for future treatment of mitochondrial liver disease.

Dual Layer Mixed Matrix Hollow Fiber Membranes for Outside-In Filtration of Human Blood Plasma

Hemodialysis mainly removes small water-soluble uremic toxins but cannot effectively remove middle molecules and protein-bound uremic toxins. Besides, the therapy is intermittent leading to fluctuating blood values and fluid status which adversely impacts patients’ health. Prolonged hemodialysis could improve the removal of toxins and the development of portable and wearable artificial kidneys could offer more flexibility in the dialysis scheme. This would enhance patients’ overall health, autonomy, mobility and flexibility, allowing patients to participate in social and economic life. However, during prolonged hemodialysis, blood clots could obstruct the fiber lumen, resulting in a decrease of the effective membrane surface area available for toxin removal. The outside-in filtration (OIF) mode, wherein blood flows through the inter-fiber space instead of through the fiber lumina, has been applied widely in blood oxygenators to prevent fiber clotting, but not in hemodialysis. In this study, we present for the first time the development of a mixed matrix membrane (MMM) for OIF of human blood plasma. This MMM combines diffusion and adsorption and consists of a polymeric membrane matrix with activated carbon (AC) particles on the inside layer, and a polymeric particle-free layer on the outer fiber layer. Our results show that in vitro MMM fibers for OIF demonstrate superior removal of the protein-bound uremic toxins, indoxyl sulfate and hippuric acid, compared to both earlier MMM fibers designed for inside-out filtration mode and commercial high-flux fibers.

A tube-in-tube membrane microreactor for tertiary treatment of urban wastewaters by photo-Fenton at neutral pH: A proof of concept

This work presents a disruptive approach to promote highly-efficient photo-Fenton process at neutral pH under continuous mode operation. The system consists of a tube-in-tube membrane reactor designed for continuous-flow titration of low iron doses to the annular reaction zone (ARZ). A concentrated acidic ferrous ion (Fe2+) solution is fed by the lumen-side of the membrane, permeating through the membrane pores (inside-out mode), being dosed and uniformly delivered to the membrane shell-side. Polluted water, containing amoxicillin (AMX) and oxidant (H2O2), flows continuously in the reactor annulus (space between the membrane shell-side and an outer quartz tube). The catalyst radial dispersion is enhanced by the helicoidal movement of water around the membrane shell-side, efficiently promoting its contact with H2O2 and UV light. The efficiency of photochemical and photocatalytic oxidation was evaluated as a function of catalyst dose, catalyst injection mode (radial permeation vs injection upstream from the reactor inlet), light source (UVA vs UVC) and aqueous solution matrix (synthetic vs real wastewater). At steady-state, photo-Fenton reaction with Fe2+ radial addition, driven by UVC light, showed the highest AMX removal for synthetic (∼65%, removal rate of 44 μMAMX/min, using [Fe2+]ARZ = 2 mg/L and [H2O2]inlet = 10 mg/L) and real municipal wastewaters (∼45%, removal rate of 31 μMAMX/min, with [Fe2+]ARZ = 5 mg/L and [H2O2]inlet = 40 mg/L), with a residence time of only 4.6 s.

Heterogeneous photocatalytic degradation of pharmaceuticals in synthetic and real matrices using a tube-in-tube membrane reactor with radial addition of H2O2

A tube-in-tube membrane reactor, with radial addition of hydrogen peroxide, was used for the oxidation of four pharmaceuticals, paracetamol (PCT), furosemide (FRS), nimesulide (NMD), and diazepam (DZP), in a continuous-mode operation, using photochemical and photocatalytic processes, driven by UVA or UVC photons. This reactor allows a controlled titration of small H2O2 doses (inside-out mode) to the catalyst particles immobilized in the membrane shell side and to the annular space between the membrane inner tubing and the concentric outer quartz tubing, where water to be treated flows. Tests were performed using synthetic (SWW) and real (urban wastewater after secondary treatment) (UWW) matrices, both spiked with the pharmaceutical mix solution (200 μg L−1 of each). The photochemical and photocatalytic oxidation efficiency was evaluated as a function of H2O2 dose (5–20 mg L−1), oxidant injection mode (radial permeation vs injection upstream from the reactor inlet), light source (UVA vs UVC lamps) and aqueous matrix (synthetic vs real matrix). At steady-state regime, the UVC/H2O2/TiO2 system, with radial H2O2 addition (20 mg L−1), showed the highest pharmaceuticals removal percentage, PCT (27.4%), FRS (35.0%), NMD (24.2%) and DZP (30.0%) in SWW. A substantial decrease in pharmaceuticals elimination was observed for UWW (PCT - 11.5%, FRS - 20.3%, NMD - 8.2% and DZP - 12.6%), in comparison with the SWW matrix. Finally, twelve transformation products (TPs) were identified; most of them showed in their structures hydroxylation in aromatic moiety; all TPs chemical structures were evaluated by BIOWIN software indicating that the TPs are non-biodegradables.

SDSS-IV MaNGA: Excavating the fossil record of stellar populations in spiral galaxies

ABSTRACT We perform a ‘fossil record’ analysis for ≈800 low-redshift spiral galaxies, using starlight applied to integral field spectroscopic observations from the SDSS-IV MaNGA survey to obtain fully spatially resolved high-resolution star formation histories (SFHs). From the SFHs, we are able to build maps indicating the present-day distribution of stellar populations of different ages in each galaxy. We find small negative mean age gradients in most spiral galaxies, especially at high stellar mass, which reflects the formation times of stellar populations at different galactocentric radii. We show that the youngest (<108.5 yr) populations exhibit significantly more extended distributions than the oldest (>109.5 yr), again with a strong dependence on stellar mass. By interpreting the radial profiles of ‘time slices’ as indicative of the size of the galaxy at the time those populations had formed, we are able to trace the simultaneous growth in mass and size of the spiral galaxies over the last 10 Gyr. Despite finding that the evolution of the measured light-weighted radius is consistent with inside-out growth in the majority of spiral galaxies, the evolution of an equivalent mass-weighted radius has changed little over the same time period. Since radial migration effects are likely to be small, we conclude that the growth of discs in spiral galaxies has occurred predominantly through an inside-out mode (with the effect greatest in high-mass galaxies), but this has not had anywhere near as much impact on the distribution of mass within spiral galaxies.

Identification of a PEST Sequence in Vertebrate KIR2.1 That Modifies Rectification.

KIR2.1 potassium channels, producing inward rectifier potassium current (IK1), are important for final action potential repolarization and a stable resting membrane potential in excitable cells like cardiomyocytes. Abnormal KIR2.1 function, either decreased or increased, associates with diseases such as Andersen-Tawil syndrome, long and short QT syndromes. KIR2.1 ion channel protein trafficking and subcellular anchoring depends on intrinsic specific short amino acid sequences. We hypothesized that combining an evolutionary based sequence comparison and bioinformatics will identify new functional domains within the C-terminus of the KIR2.1 protein, which function could be determined by mutation analysis. We determined PEST domain signatures, rich in proline (P), glutamic acid (E), serine (S), and threonine (T), within KIR2.1 sequences using the “epestfind” webtool. WT and ΔPEST KIR2.1 channels were expressed in HEK293T and COS-7 cells. Patch-clamp electrophysiology measurements were performed in the inside-out mode on excised membrane patches and the whole cell mode using AxonPatch 200B amplifiers. KIR2.1 protein expression levels were determined by western blot analysis. Immunofluorescence microscopy was used to determine KIR2.1 subcellular localization. An evolutionary conserved PEST domain was identified in the C-terminus of the KIR2.1 channel protein displaying positive PEST scores in vertebrates ranging from fish to human. No similar PEST domain was detected in KIR2.2, KIR2.3, and KIR2.6 proteins. Deletion of the PEST domain in California kingsnake and human KIR2.1 proteins (ΔPEST), did not affect plasma membrane localization. Co-expression of WT and ΔPEST KIR2.1 proteins resulted in heterotetrameric channel formation. Deletion of the PEST domain did not increase protein stability in cycloheximide assays [T½ from 2.64 h (WT) to 1.67 h (ΔPEST), n.s.]. WT and ΔPEST channels, either from human or snake, produced typical IK1, however, human ΔPEST channels displayed stronger intrinsic rectification. The current observations suggest that the PEST sequence of KIR2.1 is not associated with rapid protein degradation, and has a role in the rectification behavior of IK1 channels.

Abstract P252: Angiotensin II Type 1 Receptor Autoantibody Inhibited Bk Channels In Vascular Smooth Muscle Cells

Aims: The angiotensin II type 1 receptor autoantibody (AT1-AA) leads pathological sustainable vasoconstriction. Large-conductance Ca 2+ -and voltage-activated potassium (BK) channel, mainly consist by functional α subunits and modulatory β1 subunits in vascular smooth muscle cells (SMCs), plays a vital role on vascular relaxation. Recently, it has been found that AT1-AA inhibited BK channel’s function. However, the specific mechanisms remain unclear. In this study, we further investigated the open probability, Ca 2+ sensitivity and β1 subunits of BK channel after AT1-AA stimulation. Methods and Results: BKα subunit has several splicing variants. Immunofluorescence and PCR results showed that BK channels widely distributed among mesenteric artery (MA), most of them belong to zero-type. SMCs from MA were isolated freshly and used for patch clamp within 6 hours. AT1-AA downregulated the open probability of BK channel to 57.41±21.36% (the corresponding data of baseline was considered as 100%), specifically reduced the number of simultaneous opened channels (level 4 to level 2) instead of dwell time (T 1 =0.45 ms, T 2 = 3.39 ms to T 1 =0.58 ms, T 2 =4.99 ms) and amplitude (187.4 ±16.23 to 187.3 ±13.80 pS). This phenomenon could be reversed by BK channel agonist (NS1619) (enhanced to 124%). Noteworthy, different from Ang II, AT1-AA suppressed BK channel’s open probability consistently even after 15min (Ang II: recovered to 164.8%; AT1-AA, kept at 66.0%). The activated effect of Ca 2+ on BK channel nearly disappeared after bathing with AT1-AA in inside-out recording mode (vehicle: 100% to 471.6%; AT1-AA: 100% to 163.9%). Furthermore, to explore whether AT1-AA’s inhibitory effect on BK channel was independent from β1 subunit, only ZERO-BKα-GFP subunits were transfected into HEK293T cells. AT1-AA still diminished BK channels’ function to 57.35±5.4%. However, the inhibition of AT1-AA on BK channels was almost non-existent (to 92.1±12.81%) in AT1 receptor knock out rats. Conclusion: These results demonstrated that AT1-AA sustainably decreased open probability and Ca 2+ sensitivity of BK channels even if β1 subunit were absence. This effect was AT1 receptor dependent. It remains us that BK channel may become a novel target for improving AT1-AA related hypertension.

Physiological and pharmacological characterization of the N1303K mutant CFTR

BackgroundN1303K, one of the common, severe disease-causing mutations in the CFTR gene, causes both defective biogenesis and gating abnormalities of the CFTR protein. The goals of the present study are to quantitatively assess the gating defects associated with the N1303K mutation and its pharmacological response to CFTR modulators including potentiators VX-770 and GLPG1837 and correctors VX-809, and VX-661. MethodsGating behavior and pharmacological responses to CFTR potentiators were assessed using patch-clamp technique in the excised, inside-out mode. We also examined the effects of GLPG1837, VX-770, VX-809 and VX-661 on N1303K-CFTR surface expression using Western blot analysis. ResultsLike wild-type (WT) CFTR, N1303K-CFTR channels were activated by protein kinase A-dependent phosphorylation, but the open probability (Po) of phosphorylated N1303K-CFTR was extremely low (~0.03 vs ~0.45 in WT channels). N1303K mutants showed abnormal responses to ATP analogs or mutations that disrupt ATP hydrolysis and/or dimerization of CFTR's two nucleotide-binding domains (NBDs). However, the Po of N1303K-CFTR was dramatically increased by GLPG1837 (~17-fold) and VX-770 (~8-fold). VX-809 or VX-661 enhanced N1303K-CFTR maturation by 2–3 fold, and co-treatment with GLPG1837 or VX-770 did not show any negative drug-drug interaction. ConclusionN1303K has a severe gating defect, reduced ATP-dependence and aberrant response to ATP analogs. These results suggest a defective function of the NBDs in N1303K-CFTR. An improvement of channel function by GLPG1837 or VX-770 and an increase of Band C protein by VX-809 or VX-661 support a therapeutic strategy of combining CFTR potentiator and corrector for patients carrying the N1303K mutation.

Outsiders looking in and insiders looking out: A comparative study of newspaper coverage of Italian-German relationships in the 2013 elections

ABSTRACTWe conceptualise transnational political communication as an emerging phenomenon in globally connected polities, whereby public discourse in a country increasingly focuses on politics, policies, and political actors in other countries. There are two dimensions to transnationalisation: ‘outside-in’, when political communication focuses on the domestic politics of foreign countries, and ‘inside-out’, when foreign countries play a role in a country’s domestic public discourse. We apply this conceptual distinction to a study of newspaper coverage of the 2013 German and Italian elections in both countries. Based on a content analysis of 428 articles across six newspapers, we find that Germany received more, and more positive, coverage in the Italian press than Italy did in the German press. While the German press tended to be neutral and to cover Italy only as part of Italy’s own electoral politics, the Italian press intensely covered Germany during both the German and Italian elections. Articles in the inside-out mode of transnationalisation were more likely to discuss the interdependencies between the two countries, but those articles could only be found in the less powerful Italy, whereas they were nearly absent in the more powerful Germany.

PA-6 inhibits inward rectifier currents carried by V93I and D172N gain-of-function KIR2.1 channels, but increases channel protein expression

BackgroundThe inward rectifier potassium current IK1 contributes to a stable resting membrane potential and phase 3 repolarization of the cardiac action potential. KCNJ2 gain-of-function mutations V93I and D172N associate with increased IK1, short QT syndrome type 3 and congenital atrial fibrillation. Pentamidine-Analogue 6 (PA-6) is an efficient (IC50 = 14 nM with inside-out patch clamp methodology) and specific IK1 inhibitor that interacts with the cytoplasmic pore region of the KIR2.1 ion channel, encoded by KCNJ2. At 10 μM, PA-6 increases wild-type (WT) KIR2.1 expression in HEK293T cells upon chronic treatment. We hypothesized that PA-6 will interact with and inhibit V93I and D172N KIR2.1 channels, whereas impact on channel expression at the plasma membrane requires higher concentrations.MethodsMolecular modelling was performed with the human KIR2.1 closed state homology model using FlexX. WT and mutant KIR2.1 channels were expressed in HEK293 cells. Patch-clamp single cell electrophysiology measurements were performed in the whole cell and inside-out mode of the patch clamp method. KIR2.1 expression level and localization were determined by western blot analysis and immunofluorescence microscopy, respectively.ResultsPA-6 docking in the V93I/D172N double mutant homology model of KIR2.1 demonstrated that mutations and drug-binding site are >30 Å apart. PA-6 inhibited WT and V93I outward currents with similar potency (IC50 = 35.5 and 43.6 nM at +50 mV for WT and V93I), whereas D172N currents were less sensitive (IC50 = 128.9 nM at +50 mV) using inside-out patch-clamp electrophysiology. In whole cell mode, 1 μM of PA-6 inhibited outward IK1 at −50 mV by 28 ± 36%, 18 ± 20% and 10 ± 6%, for WT, V93I and D172N channels respectively. Western blot analysis demonstrated that PA-6 (5 μM, 24 h) increased KIR2.1 expression levels of WT (6.3 ± 1.5 fold), and V93I (3.9 ± 0.9) and D172N (4.8 ± 2.0) mutants. Immunofluorescent microscopy demonstrated dose-dependent intracellular KIR2.1 accumulation following chronic PA-6 application (24 h, 1 and 5 μM).Conclusions1) KCNJ2 gain-of-function mutations V93I and D172N in the KIR2.1 ion channel do not impair PA-6 mediated inhibition of IK1, 2) PA-6 elevates KIR2.1 protein expression and induces intracellular KIR2.1 accumulation, 3) PA-6 is a strong candidate for further preclinical evaluation in treatment of congenital SQT3 and AF.

SDSS IV MaNGA: the global and local stellar mass assemby histories of galaxies

By means of the fossil record method implemented through Pipe3D, we reconstruct the global and radial stellar mass growth histories (MGHs) of an unprecedentedly large sample of galaxies, ranging from dwarf to giant objects, from the "Mapping Nearby Galaxies at the Apache Point Observatory" survey. We confirm that the main driver of the global MGHs is mass, with more massive galaxies assembling their masses earlier (downsizing), though for a given mass, the global MGHs segregate by color, specific star formation rate (sSFR), and morphological type. From the inferred radial mean MGHs, we find that at the late evolutionary stages (or for fractions of assembled mass larger than ~ 80%), the innermost regions formed stars on average earlier than the outermost ones (inside-out). At earlier epochs, when the age resolution of the method becomes poor, the mass assembly seems to be spatially homogeneous or even in the outside-in mode, specially for the red/quiescent/early-type galaxies. The innermost MGHs are in general more regular (less scatter around the mean) than the outermost ones. For dwarf and low-mass galaxies, we do not find evidence of an outside-in formation mode; instead their radial MGHs are very diverse most of the time, with periods of outside- in and inside-out modes (or strong radial migration), suggesting this an episodic SF history. Blue/star-forming/late-type galaxies present on average a significantly more pronounced inside-out formation mode than red/quiescent/early-type galaxies, independently of mass. We discuss our results in the light of the processes of galaxy formation, quenching, and radial migration. We discuss also on the uncertainties and biases of the fossil record method and how they could affect our results.

PKA and phosphatases attached to the CaV1.2 channel regulate channel activity in cell-free patches

Calmodulin (CaM) + ATP can reprime voltage-gated L-type Ca(2+) channels (Ca(V)1.2) in inside-out patches for activation, but this effect decreases time dependently. This suggests that the Ca(V)1.2 channel activity is regulated by additional cytoplasmic factors. To test this hypothesis, we examined the role of cAMP-dependent protein kinase A (PKA) and protein phosphatases in the regulation of Ca(V)1.2 channel activity in the inside-out mode in guinea pig ventricular myocytes. Ca(V)1.2 channel activity quickly disappeared after the patch was excised from the cell and recovered to only 9% of that in the cell-attached mode on application of CaM + ATP at 10 min after the inside out. However, immediate exposure of the excised patch to the catalytic subunit of PKA + ATP or the nonspecific phosphatase inhibitor okadaic acid significantly increased the Ca(V)1.2 channel activity recovery by CaM + ATP (114 and 96%, respectively) at 10 min. Interestingly, incubation of the excised patches with cAMP + ATP also increased CaM/ATP-induced Ca(V)1.2 channel activity recovery (108%), and this effect was blocked by the nonspecific protein kinase inhibitor K252a. The channel activity in the inside-out mode was not maintained by either catalytic subunit of PKA or cAMP + ATP in the absence of CaM, but was stably maintained in the presence of CaM for more than 40 min. These results suggest that PKA and phosphatase(s) attached on or near the Ca(V)1.2 channel regulate the basal channel activity, presumably through modulation of the dynamic CaM interaction with the channel.

Regulation of the Cav1.2 cardiac channel by redox via modulation of CaM interaction with the channel

Although it has been well documented that redox can modulate Cav1.2 channel activity, the underlying mechanisms are not fully understood. In our study, we examined the effects of redox on Cav1.2 channel activity and on CaM interaction with the Cav1.2 α1 subunit. Dithiothreitol (DTT, 1 mM) in the cell-attached mode decreased, while hydrogen peroxide (H2O2, 1 mM) increased channel activity to 72 and 303%, respectively. The effects of redox were maintained in the inside-out mode where channel activity was induced by CaM + ATP: DTT (1 mM) decreased, while H2O2 (1 mM) increased the channel activity. These results were mimicked by the thioredoxin and oxidized glutathione system. To test whether the redox state might determine channel activity by affecting the CaM interaction with the channel, we examined the effects of DTT and H2O2 on CaM binding to the N- and C-terminal fragments of the channel. We found that DTT concentration-dependently inhibited CaM binding to the C-terminus (IC50 37 μM), but H2O2 had no effect. Neither DTT nor H2O2 had an effect on CaM interaction with the N-terminus. These results suggest that redox-mediated regulation of the Cav1.2 channel is governed, at least partially, by modulation of the CaM interaction with the channel.

Nucleotides maintain the activity of Cav1.2 channels in guinea-pig ventricular myocytes

The activity of Cav1.2 Ca2+ channels is maintained in the presence of calmodulin and ATP, even in cell-free patches, and thus a channel ATP-binding site has been suggested. In this study, we examined whether other nucleotides, such as GTP, UTP, CTP, ADP and AMP, could be substituted for ATP in guinea-pig ventricular myocytes. We found that all the nucleotides tested could re-prime the Ca2+ channels in the presence of 1 μM calmodulin in the inside-out mode. The order of efficacy was ATP > GTP > UTP > ADP > CTP ≈ AMP. Thus, the presumed nucleotide-binding site in the channel seemed to favor a purine rather than pyrimidine base and a triphosphate rather than a di- or mono-phosphate group. Furthermore, a high concentration (10 mM) of GTP, UTP, CTP, ADP and AMP had inhibitory effects on the channel activity. These results provide information on the putative nucleotide-binding site(s) in Cav1.2 Ca2+ channels.

Mechanosensitive Ca2 +-permeable channels in human leukemic cells: Pharmacological and molecular evidence for TRPV2

Mechanosensitive channels are present in almost every living cell, yet the evidence for their functional presence in T lymphocytes is absent. In this study, by means of the patch-clamp technique in attached and inside-out modes, we have characterized cationic channels, rapidly activated by membrane stretch in Jurkat T lymphoblasts. The half-activation was achieved at a negative pressure of ~50mm Hg. In attached mode, single channel currents displayed an inward rectification and the unitary conductance of ~40 pS at zero command voltage. In excised inside-out patches the rectification was transformed to an outward one. Mechanosensitive channels weakly discriminated between mono- and divalent cations (PCa/PNa~1) and were equally permeable for Ca2+ and Mg2+. Pharmacological analysis showed that the mechanosensitive channels were potently blocked by amiloride (1mM) and Gd3+ (10μM) in a voltage-dependent manner. They were also almost completely blocked by ruthenium red (1μM) and SKF 96365 (250μM), inhibitors of transient receptor potential vanilloid 2 (TRPV2) channels. At the same time, the channels were insensitive to 2-aminoethoxydiphenyl borate (2-APB, 100μM) or N-(p-amylcinnamoyl)anthranilic acid (ACA, 50μM), antagonists of transient receptor potential canonical (TRPC) or transient receptor potential melastatin (TRPM) channels, respectively. Human TRPV2 siRNA virtually abolished the stretch-activated current. TRPV2 are channels with multifaceted functions and regulatory mechanisms, with potentially important roles in the lymphocyte Ca2+ signaling. Implications of their regulation by mechanical stress are discussed in the context of lymphoid cells functions.