Mysterious Ca2+-independent muscular contraction: déjà vu

2012 ◽  
Vol 445 (3) ◽  
pp. 333-336 ◽  
Author(s):  
Andrey V. Kuznetsov ◽  
Rita Guzun ◽  
François Boucher ◽  
Rafaela Bagur ◽  
Tuuli Kaambre ◽  
...  
Keyword(s):  
Elevated Temperatures ◽  
Muscular Contraction ◽  
Regulation Of Respiration ◽  
Muscle Fibres ◽  
Permeabilized Cells ◽  
Functional Parameters ◽  
Respiration Of Mitochondria ◽  
Muscle Contractile ◽  
Permeabilized Muscle

The permeabilized cells and muscle fibres technique allows one to study the functional properties of mitochondria without their isolation, thus preserving all of the contacts with cellular structures, mostly the cytoskeleton, to study the whole mitochondrial population in the cell in their natural surroundings and it is increasingly being used in both experimental and clinical studies. The functional parameters (affinity for ADP in regulation of respiration) of mitochondria in permeabilized myocytes or myocardial fibres are very different from those in isolated mitochondria in vitro. In the present study, we have analysed the data showing the dependence of this parameter upon the muscle contractile state. Most remarkable is the effect of recently described Ca2+-independent contraction of permeabilized muscle fibres induced by elevated temperatures (30–37°C). We show that very similar strong spontaneous Ca2+-independent contraction can be produced by proteolytic treatment of permeabilized muscle fibres that result in a disorganization of mitochondrial arrangement, leading to a significant increase in affinity for ADP. These data show that Ca2+-insensitive contraction may be related to the destruction of cytoskeleton structures by intracellular proteases. Therefore the use of their inhibitors is strongly advised at the permeabilization step with careful washing of fibres or cells afterwards. A possible physiologically relevant relationship between Ca2+-regulated ATP-dependent contraction and mitochondrial functional parameters is also discussed.

Effect of Temperature, Velocity Gradient and I.V. Heparin on in Vitro Blood Coagulation and Platelet Aggregation

1967 ◽  
Vol 17 (01/02) ◽  
pp. 112-119 ◽  
Author(s):  
L Dintenfass ◽  
M. C Rozenberg
Keyword(s):  
Blood Coagulation ◽  
Velocity Gradient ◽  
Elevated Temperatures ◽  
Morphological Changes ◽  
Effect Of Temperature ◽  
Clotting Time ◽  
Progressive Change ◽  
Aggregation Of Platelets ◽  
Microscopic Techniques

SummaryA study of blood coagulation was carried out by observing changes in the blood viscosity of blood coagulating in the cone-in-cone viscometer. The clots were investigated by microscopic techniques.Immediately after blood is obtained by venepuncture, viscosity of blood remains constant for a certain “latent” period. The duration of this period depends not only on the intrinsic properties of the blood sample, but also on temperature and rate of shear used during blood storage. An increase of temperature decreases the clotting time ; also, an increase in the rate of shear decreases the clotting time.It is confirmed that morphological changes take place in blood coagula as a function of the velocity gradient at which such coagulation takes place. There is a progressive change from the red clot to white thrombus as the rates of shear increase. Aggregation of platelets increases as the rate of shear increases.This pattern is maintained with changes of temperature, although aggregation of platelets appears to be increased at elevated temperatures.Intravenously added heparin affects the clotting time and the aggregation of platelets in in vitro coagulation.

scholarly journals In vitro and in vivo applications of Euphorbia wallichii shoot extract-mediated gold nanospheres

2021 ◽  
Vol 10 (1) ◽  
pp. 101-111
Author(s):  
Rehman Ullah ◽  
Sumaira Shah ◽  
Zahir Muhammad ◽  
Sajjad Ali Shah ◽  
Shah Faisal ◽  
...  
Keyword(s):  
Gold Nanoparticles ◽  
Muscular Contraction ◽  
Surface Plasmon Resonance Peak ◽  
Plasmon Resonance Peak ◽  
Optical Absorption Peak ◽  
Visible Spectra ◽  
Scattering Spectra ◽  
Dose Dependent

Abstract The current study was designed to investigate the potential of Euphorbia wallichii shoot extract for reducting Au3+ and stabilizing gold nanoparticles. UV-visible spectra of gold nanoparticles showed obvious surface plasmon resonance peak at 548 nm. Microscopy (SEM and TEM) showed spherical dimensions, and the energy dispersive X-ray spectra displayed the strongest optical absorption peak for gold (Au) at 2.1 keV. Dynamic light scattering spectra represent polydispersed mixture with particulate diameter of 2.5–103.2 nm. The IR spectra confirm the potential functional groups of shoot extract responsible for the reduction of Au3+ to gold nanoparticles which exhibit tremendous antibacterial potential of 76.31%, 68.47%, 79.85%, 48.10%, and 65.53% against Escherichia coli, Staphylococcus aureus, Bacillus pumilus, Pseudomonas aeruginosa, and Klebsiella pneumoniae, respectively. Gold nanoparticles showed markedly elevated fungicidal potency compared to the shoot extract alone against the tested fungal strains. IC50 for 2,2-diphenyl-1-picrylhydrazyl scavenging was 31.52, 18.29, and 15.32 µg/mL at 30, 60, and 90 min of reaction time, respectively. Both shoot extract and nanoparticles revealed 71% mortality at 100 µg/mL, with LD90 values of 310.56 µg/mL. Experimental mice acquired dose-dependent analgesia of 54.21%, 82.60%, and 86.53% when treated with gold nanoparticles at 50, 100, and 200 mg/kg bw. Inhibition of gastrointestinal muscular contraction was 21.16%, 30.49%, and 40.19% in mice feed with 50, 100, and 200 mg/kg bw, respectively.

scholarly journals Visualization and Molecular Analysis of Actin Assembly in Living Cells

1998 ◽  
Vol 143 (7) ◽  
pp. 1919-1930 ◽  
Author(s):  
Dorothy A. Schafer ◽  
Matthew D. Welch ◽  
Laura M. Machesky ◽  
Paul C. Bridgman ◽  
Shelley M. Meyer ◽  
...  
Keyword(s):  
Actin Filament ◽  
Eukaryotic Cell ◽  
Cell Periphery ◽  
Actin Assembly ◽  
Cortical Actin ◽  
Lim Kinase ◽  
Permeabilized Cells ◽  
Capping Protein ◽  
Filament Assembly

Actin filament assembly is critical for eukaryotic cell motility. Arp2/3 complex and capping protein (CP) regulate actin assembly in vitro. To understand how these proteins regulate the dynamics of actin filament assembly in a motile cell, we visualized their distribution in living fibroblasts using green flourescent protein (GFP) tagging. Both proteins were concentrated in motile regions at the cell periphery and at dynamic spots within the lamella. Actin assembly was required for the motility and dynamics of spots and for motility at the cell periphery. In permeabilized cells, rhodamine-actin assembled at the cell periphery and at spots, indicating that actin filament barbed ends were present at these locations. Inhibition of the Rho family GTPase rac1, and to a lesser extent cdc42 and RhoA, blocked motility at the cell periphery and the formation of spots. Increased expression of phosphatidylinositol 5-kinase promoted the movement of spots. Increased expression of LIM–kinase-1, which likely inactivates cofilin, decreased the frequency of moving spots and led to the formation of aggregates of GFP–CP. We conclude that spots, which appear as small projections on the surface by whole mount electron microscopy, represent sites of actin assembly where local and transient changes in the cortical actin cytoskeleton take place.

THE INTERACTION OF ENZYMES AND HORMONES

PEDIATRICS ◽  
1960 ◽  
Vol 26 (3) ◽  
pp. 476-481
Author(s):  
Abraham White
Keyword(s):  
Direct Effect ◽  
Chemical Reactions ◽  
Elevated Temperatures ◽  
Enzyme System ◽  
Enzymic Activity ◽  
Hormone Interaction ◽  
Indirect Influence ◽  
Rare Exception ◽  
Hormonal Action

The majority of living forms depend for their functioning upon two classes of biocatalysts, the enzymes and the hormones. These biocatalysts permit the diverse chemical reactions of the organism to proceed at 38°C with a specificity and at rates frequently unattainable in vitro at elevated temperatures with similar reactants. The physiologic importance of enzymes and hormones is evident not only under normal circumstances, but is reflected clinically in the diverse descriptions of errors of metabolism, due to lack or deficiency of one or more enzymes, and the numerous hypo- and hyperfunctioning states resulting from imbalance of hormonal supply. Inasmuch as both enzymes and hormones function, with rare exception, to accelerate the rates of processes in cells, investigators have sought possible interrelationships and interactions of enzymes and hormones, particularly as a basis for the mechanism of hormonal action. It has seemed logical to hypothesize that hormones, while not essential for reactions to proceed but nevertheless affecting the rates of reactions, may function by altering either the concentration or activity of the prime cellular catalysts, the enzymes. This proposed influence of hormones on enzymic activity might be a primary, direct effect achieved by the hormone participating as an integral part of an enzyme system, or an indirect influence based upon the hormone altering the concentration of available enzyme and/or substrate utilized by a particular enzyme. It is the purpose of this presentation to describe a relatively few, but better defined, examples of the more direct relationships of enzymes and hormones. Five examples of enzyme-hormone interaction will be presented, based on the criterion that an effect of the hormone has been demonstrated on addition of the hormone in vitro to a purifled, or partially purified, enzyme system.

scholarly journals The human H5N1 influenza A virus polymerase complex is active in vitro over a broad range of temperatures, in contrast to the WSN complex, and this property can be attributed to the PB2 subunit

2008 ◽  
Vol 89 (12) ◽  
pp. 2923-2932 ◽  
Author(s):  
Birgit G. Bradel-Tretheway ◽  
Z. Kelley ◽  
Shikha Chakraborty-Sett ◽  
Toru Takimoto ◽  
Baek Kim ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Influenza A Virus ◽  
Influenza A ◽  
Elevated Temperatures ◽  
Expression System ◽  
Lung Epithelial Cells ◽  
Upper Respiratory Tract ◽  
Polymerase Complex ◽  
H5n1 Influenza

Influenza A virus (IAV) replicates in the upper respiratory tract of humans at 33 °C and in the intestinal tract of birds at close to 41 °C. The viral RNA polymerase complex comprises three subunits (PA, PB1 and PB2) and plays an important role in host adaptation. We therefore developed an in vitro system to examine the temperature sensitivity of IAV RNA polymerase complexes from different origins. Complexes were prepared from human lung epithelial cells (A549) using a novel adenoviral expression system. Affinity-purified complexes were generated that contained either all three subunits (PA/PB1/PB2) from the A/Viet/1203/04 H5N1 virus (H/H/H) or the A/WSN/33 H1N1 strain (W/W/W). We also prepared chimeric complexes in which the PB2 subunit was exchanged (H/H/W, W/W/H) or substituted with an avian PB2 from the A/chicken/Nanchang/3-120/01 H3N2 strain (W/W/N). All complexes were functional in transcription, cap-binding and endonucleolytic activity. Complexes containing the H5N1 or Nanchang PB2 protein retained transcriptional activity over a broad temperature range (30–42 °C). In contrast, complexes containing the WSN PB2 protein lost activity at elevated temperatures (39 °C or higher). The E627K mutation in the avian PB2 was not required for this effect. Finally, the avian PB2 subunit was shown to confer enhanced stability to the WSN 3P complex. These results show that PB2 plays an important role in regulating the temperature optimum for IAV RNA polymerase activity, possibly due to effects on the functional stability of the 3P complex.

scholarly journals Characterizing a thermostable Cas9 for bacterial genome editing and silencing

2017 ◽  
Author(s):  
Ioannis Mougiakos ◽  
Prarthana Mohanraju ◽  
Elleke F. Bosma ◽  
Valentijn Vrouwe ◽  
Max Finger Bou ◽  
...  
Keyword(s):  
Genome Editing ◽  
Genome Engineering ◽  
Gene Deletion ◽  
Elevated Temperatures ◽  
Bacterial Genome ◽  
Thermophilic Bacterium ◽  
Geobacillus Thermodenitrificans ◽  
Temperature Range ◽  
Fundamental Research

AbstractCRISPR-Cas9 based genome engineering tools have revolutionized fundamental research and biotechnological exploitation of both eukaryotes and prokaryotes. However, the mesophilic nature of the established Cas9 systems does not allow for applications that require enhanced stability, including engineering at elevated temperatures. Here, we identify and characterize ThermoCas9: an RNA-guided DNA-endonuclease from the thermophilic bacterium Geobacillus thermodenitrificans T12. We show that ThermoCas9 is active in vitro between 20°C and 70°C, a temperature range much broader than that of the currently used Cas9 orthologues. Additionally, we demonstrate that ThermoCas9 activity at elevated temperatures is strongly associated with the structure of the employed sgRNA. Subsequently, we develop ThermoCas9-based engineering tools for gene deletion and transcriptional silencing at 55°C in Bacillus smithii and for gene deletion at 37°C in Pseudomonas putida. Altogether, our findings provide fundamental insights into a thermophilic CRISPR-Cas family member and establish the first Cas9-based bacterial genome editing and silencing tool with a broad temperature range.

Effect of transfection and co-incubation of bovine sperm with exogenous DNA on sperm quality and functional parameters for its use in sperm-mediated gene transfer

Zygote ◽  
2016 ◽  
Vol 25 (1) ◽  
pp. 85-97 ◽  
Author(s):  
María Elena Arias ◽  
Esther Sánchez-Villalba ◽  
Andrea Delgado ◽  
Ricardo Felmer
Keyword(s):  
Gene Transfer ◽  
Sperm Quality ◽  
Computer Assisted ◽  
Exogenous Dna ◽  
Sperm Analysis ◽  
Functional Parameters ◽  
Transgenic Embryos ◽  
Fertilization Capacity ◽  
Motility Parameters

SummarySperm-mediated gene transfer (SMGT) is based on the capacity of sperm to bind exogenous DNA and transfer it into the oocyte during fertilization. In bovines, the progress of this technology has been slow due to the poor reproducibility and efficiency of the production of transgenic embryos. The aim of the present study was to evaluate the effects of different sperm transfection systems on the quality and functional parameters of sperm. Additionally, the ability of sperm to bind and incorporate exogenous DNA was assessed. These analyses were carried out by flow cytometry and confocal fluorescence microscopy, and motility parameters were also evaluated by computer-assisted sperm analysis (CASA). Transfection was carried out using complexes of plasmid DNA with Lipofectamine, SuperFect and TurboFect for 0.5, 1, 2 or 4 h. The results showed that all of the transfection treatments promoted sperm binding and incorporation of exogenous DNA, similar to sperm incorporation of DNA alone, without affecting the viability. Nevertheless, the treatments and incubation times significantly affected the motility parameters, although no effect on the integrity of DNA or the levels of reactive oxygen species (ROS) was observed. Additionally, we observed that transfection using SuperFect and TurboFect negatively affected the acrosome integrity, and TurboFect affected the mitochondrial membrane potential of sperm. In conclusion, we demonstrated binding and incorporation of exogenous DNA by sperm after transfection and confirmed the capacity of sperm to spontaneously incorporate exogenous DNA. These findings will allow the establishment of the most appropriate method [intracytoplasmic sperm injection (ICSI) orin vitrofertilization (IVF)] of generating transgenic embryos via SMGT based on the fertilization capacity of transfected sperm.

Address of the President Sir Alan Hodgkin, O. M. at the Anniversary Meeting, 30 November 1973

1974 ◽  
Vol 185 (1078) ◽  
Keyword(s):  
Surface Membrane ◽  
Muscular Activity ◽  
Nerve Impulse ◽  
Muscular Contraction ◽  
Single Fibre ◽  
Muscle Fibres ◽  
Constant Length ◽  
Axon Membrane ◽  
Ultrastructural Studies ◽  
Mathematical Skill

The Copley Medal is awarded to Professor A. F. Huxley, F. R. S. A. F. Huxley has made outstanding contributions to our knowledge of the nerve impulse and of the mechanism by which muscle fibres are caused to contract. Jointly with Hodgkin, he introduced the powerful method of intracellular recording from nerve cells and showed that during the propagation of an impulse the mem­brane potential reverses its sign, and does not simply fall towards zero as had been widely believed. This work - interrupted by the 1939-45 war, but later resumed - led to the proposal that the impulse arises from a transient influx of sodium ions through the axon membrane. The ‘ionic theory’ of nervous conduction was then established by a series of convincing experiments and calculations for which Huxley later shared the Nobel Prize. Huxley next turned his attention to the mechanism of muscular contraction. He equipped himself for this purpose by inventing a new type of interference microscope. In experiments on living isolated muscle fibres, Huxley showed that contraction is accompanied by a shortening of the isotropic band of each sarco­mere, while the remaining portion (the anisotropic band) retains approximately constant length. His findings complemented the important ultrastructural studies of H. E. Huxley and led them both to propose a ‘sliding filament’ mechanism as the basis of muscular motion. During further microscopic observations on the living muscle fibre, A. F. Huxley produced most striking evidence on the way in which an excitatory potential change of the surface membrane is communicated, through local tubular channels, to the interior of the fibre where it activates the contractile elements. In his most recent work, A. F. Huxley has continued to develop his single-fibre technique to resolve even finer details of the dynamic changes which occur during muscular activity. His work is characterized by a rare combination of profound theoretical insight, mathematical skill and superb technical mastery, all of which has enabled him to select problems of first-rate importance and to pursue them with outstanding success.

Loss of α-actinin-3 confers protection from eccentric contraction damage in fast-twitch EDL muscles from aged mdx dystrophic mice by reducing pathological fibre branching

2021 ◽  
Author(s):  
Leonit Kiriaev ◽  
Peter J. Houweling ◽  
Kathryn N. North ◽  
Stewart I. Head
Keyword(s):  
Morphological Changes ◽  
Eccentric Contraction ◽  
Muscle Fibres ◽  
Double Knockout ◽  
Reduction In Force ◽  
The Common ◽  
Fast Twitch ◽  
Skeletal Muscle Fibres ◽  
Dystrophic Mice

ABSTRACTThe common null polymorphism (R577X) in the ACTN3 gene is present in over 1.5 billion people worldwide and results in the absence of the protein α-actinin-3 from the Z-discs of fast-twitch skeletal muscle fibres. We have previously reported that this polymorphism is a modifier of dystrophin deficient Duchenne Muscular Dystrophy. To investigate the mechanism underlying this we use a double knockout (dk)Actn3KO/mdx (dKO) mouse model which lacks both dystrophin and sarcomere α-actinin-3. We used dKO mice and mdx dystrophic mice at 12 months (aged) to investigate the correlation between morphological changes to the fast-twitch dKO EDL and the reduction in force deficit produced by an in vitro eccentric contraction protocol. In the aged dKO mouse we found a marked reduction in fibre branching complexity that correlated with protection from eccentric contraction induced force deficit. Complex branches in the aged dKO EDL fibres (28%) were substantially reduced compared to aged mdx EDL fibres (68%) and this correlates with a graded force loss over three eccentric contractions for dKO muscles (∼35% after first contraction, ∼66% overall) compared to an abrupt drop in mdx upon the first eccentric contraction (∼73% after first contraction, ∼89% after three contractions). In dKO protection from eccentric contraction damage was linked with a doubling of SERCA1 pump density the EDL. We propose that the increased oxidative metabolism of fast-twitch glycolytic fibres characteristic of the null polymorphism (R577X) and increase in SR Ca2+ pump proteins reduces muscle fibre branching and decreases susceptibility to eccentric injury in the dystrophinopathies.

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