scholarly journals Sodium uptake and membrane excitation in Paramecium.

1979 ◽  
Vol 81 (2) ◽  
pp. 374-381 ◽  
Author(s):  
H G Hansma
Keyword(s):  
Resting Potential ◽  
Wild Type ◽  
Sodium Uptake ◽  
Membrane Excitation ◽  
Kinetics Of ◽  
Na Uptake ◽  
Potential Level

Although the phenotypes of many membrane-excitation mutants of Paramecium are best expressed in Na+-containing solutions, little is known about the role of Na+ in membrane excitation in Paramecium. By measuring 22Na fluxes, we have shown that: (a) The total cellular Na+ content is equivalent to a cytoplasmic concentration of 3--4 mM, if the Na+ concentration is uniform throughout the cell. (b) The kinetics of Na+ uptake can be divided into a saturable Na+ uptake with an apparent Km = 0.15 mM and a nonsaturable Na+ uptake seen at higher Na+ concentrations up to 20 mM. (c) The rate of Na+ uptake in high Na+ solutions is correlated with the duration of backward swimming and membrane excitation in wild type Paramecium and the mutants fast-2 and paranoiac. (d) Na+ uptake is inhibited at 4 degrees C. From these results, we postulate that Na+ uptake is faster when the membrane is depolarized than when it is at the resting potential level.

Endobrevin/VAMP-8-Mediated Dense Granule Release Is Required for Efficient Thrombus Formation in Vivo.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 1836-1836
Author(s):  
Price S. Blair ◽  
Qiansheng Ren ◽  
Gwenda J. Graham ◽  
James R. Dilks ◽  
Sidney W. Whiteheart ◽  
...  
Keyword(s):  
Vascular Injury ◽  
Thrombus Formation ◽  
Dense Granule ◽  
Wild Type ◽  
Platelet Accumulation ◽  
Induced Aggregation ◽  
Kinetics Of ◽  
Granule Release

Abstract Individuals whose platelets lack dense core or alpha-granules suffer varying degrees of abnormal bleeding, implying that granule cargo contributes to hemostasis. Despite these clinical observations, little is known regarding the effects of impaired platelet granule secretion on thrombus formation in vivo. The release of cargo from platelet granules requires a group of membrane proteins called SNAREs (Soluble NSF Attachment Protein Receptors) that mediate fusion of granule membranes to the plasma membrane and open canalicular system. Endobrevin/VAMP-8 is the primary vesicular-SNARE (v-SNARE) responsible for efficient release of dense core and a-granule contents. To evaluate the importance of VAMP-8-mediated secretion on the kinetics of thrombus formation in vivo, we measured platelet accumulation following laser-induced vascular injury in VAMP-8−/− mice. Three different phases of thrombus formation - initiation, maximal accumulation, and stabilized platelet accumulation - were tested. Analysis of initial thrombus formation from wild-type and VAMP-8−/− mice showed that average platelet accumulation in VAMP- 8−/− mice was 23% of accumulation in wild-type mice (P=0.009) at 30 sec following injury. There was a trend towards smaller maximal thrombus size in VAMP-8−/− mice, but the difference was not statistically significant (P=0.1). Average stabilized platelet accumulation at 180 sec in VAMP-8−/− mice was 40% of wild-type mice (P=0.05). Thus, thrombus formation is delayed and decreased in VAMP-8−/− mice, but not absent. Dense granule release occurs more rapidly than alpha-granule release, which does not occur for 2–3 min following laser-induced vascular injury. Agonist-induced dense granule release from VAMP-8−/− platelets is defective. To directly evaluate the role of dense granule release on the kinetics of thrombus formation, we assessed thrombus formation in the mouse model of Hermansky-Pudlak syndrome, ruby-eye, which lack dense granules. Thrombus formation following laser-induced vascular injury was nearly abolished in ruby-eye mice such that maximal platelet accumulation was 15% that of wild-type mice. In vitro, the thrombin doses required to induce irreversible aggregation in wild-type, VAMP-8−/−, and ruby-eye platelets were 25 mU, 50 mU, and 150 mU, respectively. Incubation with apyrase had little effect on thrombin-induced aggregation of VAMP-8−/− or ruby-eye platelets. In contrast, incubation of wild-type platelets with apyrase reduced their thrombin sensitivity compared to that of ruby-eye platelets. Supplementation with a substimulatory ADP concentration reversed the thrombin-induced aggregation defect in VAMP-8−/− and ruby-eye mice. Thus, defective ADP release is the primary abnormality leading to impaired aggregation in VAMP-8−/− and ruby-eye mice. Tail bleeding times were assessed in VAMP- 8−/− mice to evaluate the role of VAMP-8 in hemostasis. In contrast to ruby-eye mice, which have a markedly prolonged bleeding time, tail bleeding times in VAMP-8−/− mice were not significantly prolonged compared to those in wild-type mice. These results demonstrate the importance of VAMP-8 and dense granule release in the initial phases of thrombus formation and validate the distal platelet secretory machinery as a potential target for anti-platelet therapies.

Contribution of cysteine residue to the properties of Listeria monocytogenes listeriolysin O

2009 ◽  
Vol 55 (10) ◽  
pp. 1153-1159 ◽  
Author(s):  
Radosław Stachowiak ◽  
Jarosław Wiśniewski ◽  
Olga Osińska ◽  
Jacek Bielecki
Keyword(s):  
Listeria Monocytogenes ◽  
Hemolytic Activity ◽  
Cysteine Residue ◽  
Listeriolysin O ◽  
Wild Type ◽  
Gram Positive Bacteria ◽  
Culture Model ◽  
The Family ◽  
Kinetics Of

Listeriolysin (LLO) is the key virulence factor critical for Listeria monocytogenes pathogenesis. Listerial cytolysin belongs to the family of cholesterol-dependent cytolysins (CDCs), a group of pore-forming toxins produced by related gram-positive bacteria. Most CDCs contain a cysteine residue in the conserved undecapeptide — a sequence that is highly preserved among this group of proteins. Substitutions of cysteine do not always lead to loss of hemolytic activity, questioning the purpose of such strong conservation of this amino acid in the sequence of CDC. The properties of 3 L. monocytogenes strains, a wild type and 2 mutants expressing modified LLO within the cysteine residue, were analyzed in this work. The first of these mutants producing a toxin with cysteine to alanine substitution showed similar features to the wild type except that a thiol-reducing agent was not necessary for hemolytic activity. Another strain secreting LLO containing serine instead of cysteine exhibited strikingly different properties than the wild type. Modified toxin is independent of the reducing reagents, less stable, and shows accelerated kinetics of cytolysis in comparison with the unchanged protein. However, both mutant strains are less invasive in the cell culture model showing the important role of cysteine in L. monocytogenes virulence.

The Action Potential in Mammalian Neurons: New Perspectives

Physiology ◽  
1993 ◽  
Vol 8 (1) ◽  
pp. 42-44 ◽  
Author(s):  
O Sacchi ◽  
O Belluzzi
Keyword(s):  
Membrane Potential ◽  
Action Potential ◽  
Fast Kinetics ◽  
Numerical Reconstruction ◽  
Mammalian Neuron ◽  
Kinetics Of ◽  
Level 2 ◽  
A Current ◽  
Potential Level

Fresh ideas suggested by the first numerical reconstruction of the action potential in a mammalian neuron are 1) role of the A-current and the starting membrane potential level, 2) fast kinetics of the pericellular K+ accumulation, and 3) spike-related Ca2+ movements.

scholarly journals Oligomerization and hemolytic properties of the C-terminal domain of pyolysin, a cholesterol-dependent cytolysin

2013 ◽  
Vol 91 (2) ◽  
pp. 59-66 ◽  
Author(s):  
Lisa Pokrajac ◽  
J. Robin Harris ◽  
Naghmeh Sarraf ◽  
Michael Palmer
Keyword(s):  
Full Length ◽  
Membrane Insertion ◽  
Wild Type ◽  
Terminal Domain ◽  
Large Size ◽  
Monomer Structure ◽  
Streptolysin O ◽  
Kinetics Of ◽  
Domain 4

Pyolysin (PLO) belongs to the homologous family of the cholesterol-dependent cytolysins (CDCs), which bind to cell membranes containing cholesterol to form oligomeric pores of large size. The CDC monomer structure consists of 4 domains. Among these, the C-terminal domain 4 has been implicated in membrane binding of the monomer, while the subsequent processes of oligomerization and membrane insertion have primarily been assigned to other domains of the molecule. Recombinantly expressed or proteolytic fragments that span domain 4 of the CDCs streptolysin O and perfringolysin O bind to membranes but fail to oligomerize, and they inhibit the activity of the respective wild-type toxins. We report here that the isolated domain 4 of pyolysin (PLO-D4) not only binds to membranes but also forms oligomers with itself, as well as hybrid oligomers with the full-length toxin. As expected, the pure PLO-D4 oligomers are devoid of pore-forming activity. Surprisingly, however, within hybrid oligomers, PLO-D4 not only fails to inhibit, but even amplifies the hemolytic activity of the full-length toxin, to an extent similar to that of doubling the amount of the full-length toxin alone. We propose that this amplification may be related to the kinetics of the oligomerization reaction. Overall, our findings indicate a greater role of domain 4 in the oligomerization of CDCs than previously demonstrated.

scholarly journals RYR2 Proteins Contribute to the Formation of Ca2+ Sparks in Smooth Muscle

2004 ◽  
Vol 123 (4) ◽  
pp. 377-386 ◽  
Author(s):  
Guangju Ji ◽  
Morris E. Feldman ◽  
Kai Su Greene ◽  
Vincenzo Sorrentino ◽  
Hong-Bo Xin ◽  
...  
Keyword(s):  
Smooth Muscle ◽  
Calcium Release ◽  
Ryanodine Receptors ◽  
Wild Type ◽  
Calcium Dependent ◽  
Outward Currents ◽  
Associated Proteins ◽  
Kinetics Of ◽  
Spontaneous Transient Outward Currents

Calcium release through ryanodine receptors (RYR) activates calcium-dependent membrane conductances and plays an important role in excitation-contraction coupling in smooth muscle. The specific RYR isoforms associated with this release in smooth muscle, and the role of RYR-associated proteins such as FK506 binding proteins (FKBPs), has not been clearly established, however. FKBP12.6 proteins interact with RYR2 Ca2+ release channels and the absence of these proteins predictably alters the amplitude and kinetics of RYR2 unitary Ca2+ release events (Ca2+ sparks). To evaluate the role of specific RYR2 and FBKP12.6 proteins in Ca2+ release processes in smooth muscle, we compared spontaneous transient outward currents (STOCs), Ca2+ sparks, Ca2+-induced Ca2+ release, and Ca2+ waves in smooth muscle cells freshly isolated from wild-type, FKBP12.6−/−, and RYR3−/− mouse bladders. Consistent with a role of FKBP12.6 and RYR2 proteins in spontaneous Ca2+ sparks, we show that the frequency, amplitude, and kinetics of spontaneous, transient outward currents (STOCs) and spontaneous Ca2+ sparks are altered in FKBP12.6 deficient myocytes relative to wild-type and RYR3 null cells, which were not significantly different from each other. Ca2+ -induced Ca2+ release was similarly augmented in FKBP12.6−/−, but not in RYR3 null cells relative to wild-type. Finally, Ca2+ wave speed evoked by CICR was not different in RYR3 cells relative to control, indicating that these proteins are not necessary for normal Ca2+ wave propagation. The effect of FKBP12.6 deletion on the frequency, amplitude, and kinetics of spontaneous and evoked Ca2+ sparks in smooth muscle, and the finding of normal Ca2+ sparks and CICR in RYR3 null mice, indicate that Ca2+ release through RYR2 molecules contributes to the formation of spontaneous and evoked Ca2+ sparks, and associated STOCs, in smooth muscle.

scholarly journals Changes in Expression Level of OsHKT1;5 Alters Activity of Membrane Transporters Involved in K+ and Ca2+ Acquisition and Homeostasis in Salinized Rice Roots

2020 ◽  
Vol 21 (14) ◽  
pp. 4882
Author(s):  
Mohammad Alnayef ◽  
Celymar Solis ◽  
Lana Shabala ◽  
Takaaki Ogura ◽  
Zhonghua Chen ◽  
...  
Keyword(s):  
Direct Evidence ◽  
Xylem Sap ◽  
Feedback Regulation ◽  
Membrane Transporters ◽  
Expression Level ◽  
Xylem Parenchyma ◽  
Critical Determinant ◽  
Kinetics Of ◽  
Na Uptake

In rice, the OsHKT1;5 gene has been reported to be a critical determinant of salt tolerance. This gene is harbored by the SKC1 locus, and its role was attributed to Na+ unloading from the xylem. No direct evidence, however, was provided in previous studies. Also, the reported function of SKC1 on the loading and delivery of K+ to the shoot remains to be explained. In this work, we used an electrophysiological approach to compare the kinetics of Na+ uptake by root xylem parenchyma cells using wild type (WT) and NIL(SKC1) plants. Our data showed that Na+ reabsorption was observed in WT, but not NIL(SKC1) plants, thus questioning the functional role of HKT1;5 as a transporter operating in the direct Na+ removal from the xylem. Instead, changes in the expression level of HKT1;5 altered the activity of membrane transporters involved in K+ and Ca2+ acquisition and homeostasis in the rice epidermis and stele, explaining the observed phenotype. We conclude that the role of HKT1;5 in plant salinity tolerance cannot be attributed to merely reducing Na+ concentration in the xylem sap but triggers a complex feedback regulation of activities of other transporters involved in the maintenance of plant ionic homeostasis and signaling under stress conditions.

scholarly journals Regulatory role of the respiratory supercomplex factors in Saccharomyces cerevisiae

2016 ◽  
Vol 113 (31) ◽  
pp. E4476-E4485 ◽  
Author(s):  
Camilla Rydström Lundin ◽  
Christoph von Ballmoos ◽  
Martin Ott ◽  
Pia Ädelroth ◽  
Peter Brzezinski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Electron Transfer ◽  
Ligand Binding ◽  
Structural Changes ◽  
Regulatory Mechanism ◽  
Supramolecular Interactions ◽  
Wild Type ◽  
Single Turnover ◽  
Kinetics Of

The respiratory supercomplex factors (Rcf) 1 and 2 mediate supramolecular interactions between mitochondrial complexes III (ubiquinol-cytochrome c reductase; cyt. bc1) and IV (cytochrome c oxidase; CytcO). In addition, removal of these polypeptides results in decreased activity of CytcO, but not of cyt. bc1. In the present study, we have investigated the kinetics of ligand binding, the single-turnover reaction of CytcO with O2, and the linked cyt. bc1-CytcO quinol oxidation-oxygen-reduction activities in mitochondria in which Rcf1 or Rcf2 were removed genetically (strains rcf1Δ and rcf2Δ, respectively). The data show that in the rcf1Δ and rcf2Δ strains, in a significant fraction of the population, ligand binding occurs over a time scale that is ∼100-fold faster (τ ≅ 100 μs) than observed with the wild-type mitochondria (τ ≅ 10 ms), indicating structural changes. This effect is specific to removal of Rcf and not dissociation of the cyt. bc1–CytcO supercomplex. Furthermore, in the rcf1Δ and rcf2Δ strains, the single-turnover reaction of CytcO with O2 was incomplete. This observation indicates that the lower activity of CytcO is caused by a fraction of inactive CytcO rather than decreased CytcO activity of the entire population. Furthermore, the data suggest that the Rcf1 polypeptide mediates formation of an electron-transfer bridge from cyt. bc1 to CytcO via a tightly bound cyt. c. We discuss the significance of the proposed regulatory mechanism of Rcf1 and Rcf2 in the context of supramolecular interactions between cyt. bc1 and CytcO.

scholarly journals Oxytocin receptors influence the development and maintenance of social behavior in zebrafish (Danio rerio).

2021 ◽  
Author(s):  
Anja Gemmer ◽  
Kristina Mirkes ◽  
Lukas Anneser ◽  
Tim Eilers ◽  
Caroline Kibat ◽  
...  
Keyword(s):  
Social Behavior ◽  
Social Preference ◽  
Autism Spectrum ◽  
Social Impairment ◽  
Wild Type ◽  
Knock Out ◽  
Oxytocin Receptors ◽  
Shoaling Behavior ◽  
Kinetics Of

Zebrafish are highly social teleost fish and an excellent model to study social behavior. The neuropeptide Oxytocin is associated different social behaviors as well as disorders resulting in social impairment like autism spectrum disorder. However, how Oxytocin receptor signaling affects the development and expression kinetics of social behavior is not known. In this study we investigated the role of the two oxytocin receptors, Oxtr and Oxtrl, in the development and maintenance of social preference and shoaling behavior in 2- to 8-week-old zebrafish. Using CRISPR/Cas9 mediated oxtr and oxtrl knock-out fish, we found that the development of social preference is accelerated if one of the Oxytocin receptors is knocked-out and that the knock-out fish reach significantly higher levels of social preference. Moreover, oxtr-/- fish showed impairments in the maintenance of social preference. Social isolation prior to testing led to impaired maintenance of social preference in both wild-type and oxtr and oxtrl knock-out fish. Knocking-out one of the Oxytocin receptors also led to increased group spacing and reduced polarization in a 20-fish shoal at 8 weeks post fertilization, but not at 4. These results show that the development and maintenance of social behavior is influenced by the Oxytocin receptors and that the effects are not just pro- or antisocial, but dependent on both the age and social context of the fish.  

scholarly journals Role of TLR4 in persistent Leptospira interrogans infection: a comparative in vivo study in mice

2020 ◽  
Author(s):  
Nisha Nair ◽  
Mariana Soares Guedes ◽  
Adeline Hajjar ◽  
Catherine Werts ◽  
Maria Gomes-Solecki
Keyword(s):  
Transgenic Mice ◽  
Sublethal Dose ◽  
Wild Type ◽  
Immune Markers ◽  
Lethal Infection ◽  
Tlr4 Gene ◽  
Kinetics Of ◽  
Tlr4 Receptor

AbstractToll-Like Receptor (TLR) 4, the LPS receptor, plays a central role in the control of leptospirosis and absence of TLR4 results in lethal infection in mice. Because human TLR4 does not sense the atypical leptospiral-LPS, we hypothesized that TLR4/MD-2 humanized transgenic mice (huTLR4) may be more susceptible to leptospirosis than wild-type mice, and thus may constitute a model of acute human leptospirosis. Therefore, we infected huTLR4 mice, which express human TLR4 but not murine TLR4, with a high but sublethal dose of L. interrogans serovar Copenhageni FioCruz (Leptospira) in comparison to C57BL/6J wildtype (WT) and, as a control, a congenic strain in which the tlr4 coding sequences are deleted (muTLR4Lps-del). We show that the huTLR4 gene is fully functional in the murine background. We found that dissemination of Leptospira in blood, shedding in urine, colonization of the kidney and overall kinetics of leptospirosis progression is equivalent between WT and huTLR4 C57BL/6J mice. Furthermore, inflammation of the kidney appeared to be subdued in huTLR4 compared to WT mice in that we observed less infiltrates of mononuclear lymphocytes, less innate immune markers and no relevant differences in fibrosis markers. Contrary to our hypothesis, huTLR4 mice showed less inflammation and kidney pathology, and are not more susceptible to leptospirosis than WT mice. This study is significant as it indicates that one intact TLR4 gene, be it mouse or human, is necessary to control acute leptospirosis.Contribution to the fieldDifferences of recognition exist between mouse and human TLR4, in that the anchor of LPS in the outer membrane of Leptospira (LipidA) activates murine, but not human TLR4. We hypothesized that if human TLR4 does not sense leptospiral-LPS, then transgenic mice in which murine TLR4 was replaced with human TLR4, should be more susceptible to Leptospira dissemination as compared to congenic wild-type mice, which could result in a more robust inflammatory response and pathology in the kidney. However, we found that impaired sensing of leptospiral-LPS in huTLR4 mice did not affect overall infection in comparison to wild-type mice and does not result in increased pathology of the kidney. Our study indicates that rather than leptospiral-LPS sensing, the presence of a fully functional TLR4 receptor is necessary to control acute leptospirosis.

Protease Activated Receptor-4 (PAR4) Uses Anionic Residues to Interact with α-Thrombin in the Absence or Presence of PAR1.

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 2019-2019
Author(s):  
Marvin T Nieman
Keyword(s):  
Point Mutations ◽  
Calcium Flux ◽  
Wild Type ◽  
Individual Point ◽  
Protease Activated Receptor 1 ◽  
Anionic Cluster ◽  
Kinetics Of ◽  
Current Report

Abstract Thrombin activates protease activated receptor 1 (PAR1) faster than protease activated receptor 4 (PAR4) due to a hirudin-like sequence in the exodomain of PAR1 that binds thrombin’s exosite I. However, recombinant exodomain studies indicate that PAR4 does have extended contacts with α-thrombin that influence PAR4’s kinetics of cleavage. In the current report, the role of an anionic cluster (Asp57, Asp59, Glu62, Asp65) in the exodomain of PAR4 is examined for its influence on cleavage and activation of PAR4 on cells in the absence or presence of PAR1. α-Thrombin induces wild type PAR4 (PAR4-wt) calcium flux with an EC50 of 110 nM whereas mutation of the four anionic residues (PAR4-AAAA) increases the EC50 to 641 nM. In contrast, PAR4-wt and PAR4-AAAA are activated by γ-thrombin with a similar EC50 (588 nM and 449 nM, respectively, p = 0.48), indicating a role for α-thrombin’s exosite I in PAR4 activation. Coexpression of PAR1, lowered the EC50 of cleavage 10 fold for both PAR4-wt from 321 to 26 nM and PAR4-AAAA from 2.2 μM to 360 nM, respectively. Individual point mutations at Asp57, Asp59, Glu62 or Asp65 show that PAR4-D57A is activated by α-thrombin with the same EC50 as PAR4-wt (140 nM) whereas PAR4-D59A is the same as PAR4-AAAA (699 nM). Glu62 and Asp65 contribute to α-thrombin recognition, but to a lesser extent. The current report shows that PAR4 uses its anionic cluster to interact with α-thrombin and that this interaction is important even in the presence of PAR1.

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