Wide-field extended-resolution fluorescence microscopy with standing surface-plasmon-resonance waves

The resolution of conventional SPR imaging has been limited by the diffraction nature of light. A wide-field extended-resolution optical imaging technique, standing-wave surface plasmon resonance fluorescence (SW-SPRF) microscopy, has been developed. Based on evanescent SPR standing waves, SW-SPRF provides lateral resolution approaching 100 nm and offers the advantages of significant signal enhancement and background noise reduction. SW-SPRF has the potential for sensitive biomolecular detection, nanoscale imaging, and lithographic applications

Extraction of Stevioside from Stevia Rebaudiana Leaves Using Cellulase

Stevioside is a diterpene glycoside present in Stevia Rebaudiana leaves that has the ability to sweeten at rated between 70 to 350 times than sucrose (0.4% w/v). It has no calorific value. Unlike many low calorie sweeteners, stevioside is stable at high temperature. The objective of this research is to extract stevioside from stevia rebaudiana leaves by using cellulase from Aspergillus Niger. Acetate buffer and ethanol were used as a medium for enzyme and as a solvent, respectively. In this present study, the enzymatic extraction of stevioside from stevia rebaudiana leaves was carried out using cellulase with various parameters that affect the production of stevioside such as concentration of enzyme, incubation time and temperature. Cellulase was observed to give the highest stevioside yield (16230 ± 0.3 μg/ml) at 40oC. This indicated that the maximum temperature for cellulase activity was 40oC. The results signify that the enzymatic extraction method is an alternative to solvent based stevioside extraction, based on its higher efficiency. Thus, it can be concluded that the extraction of stevioside from Stevia rebaudiana leaves using cellulase can be maximized under the maximum conditions for the cellulase activity where the used of solvent can be minimized in degrading the cell wall Together with the maximum heat and correct combination of the solvent used, a new and efficient way of extracting high yield of stevioside can be obtained

Thermal Denaturation and Aggregation of Myosin Subfragment 1 Isoforms with Different Essential Light Chains

We compared thermally induced denaturation and aggregation of two isoforms of the isolated myosin head (myosin subfragment 1, S1) containing different “essential” (or “alkali”) light chains, A1 or A2. We applied differential scanning calorimetry (DSC) to investigate the domain structure of these two S1 isoforms. For this purpose, a special calorimetric approach was developed to analyze the DSC profiles of irreversibly denaturing multidomain proteins. Using this approach, we revealed two calorimetric domains in the S1 molecule, the more thermostable domain denaturing in two steps. Comparing the DSC data with temperature dependences of intrinsic fluorescence parameters and S1 ATPase inactivation, we have identified these two calorimetric domains as motor domain and regulatory domain of the myosin head, the motor domain being more thermostable. Some difference between the two S1 isoforms was only revealed by DSC in thermal denaturation of the regulatory domain. We also applied dynamic light scattering (DLS) to analyze the aggregation of S1 isoforms induced by their thermal denaturation. We have found no appreciable difference between these S1 isoforms in their aggregation properties under ionic strength conditions close to those in the muscle fiber (in the presence of 100 mM KCl). Under these conditions kinetics of this process was independent of protein concentration, and the aggregation rate was limited by irreversible denaturation of the S1 motor domain

Myosin Regulatory Light Chain (RLC) Phosphorylation Change as a Modulator of Cardiac Muscle Contraction in Disease

Author’s Choice—Final version full access.Understanding how cardiac myosin regulatory light chain (RLC) phosphorylation alters cardiac muscle mechanics is important because it is often altered in cardiac disease. The effect this protein phosphorylation has on muscle mechanics during a physiological range of shortening velocities, during which the heart generates power and performs work, has not been addressed. We have expressed and phosphorylated recombinant Rattus norvegicus left ventricular RLC. In vitro we have phosphorylated these recombinant species with cardiac myosin light chain kinase and zipper-interacting protein kinase. We compare rat permeabilized cardiac trabeculae, which have undergone exchange with differently phosphorylated RLC species. We were able to enrich trabecular RLC phosphorylation by 40% compared with controls and, in a separate series, lower RLC phosphorylation to 60% of control values. Compared with the trabeculae with a low level of RLC phosphorylation, RLC phosphorylation enrichment increased isometric force by more than 3-fold and peak power output by more than 7-fold and approximately doubled both maximum shortening speed and the shortening velocity that generated peak power. We augmented these measurements by observing increased RLC phosphorylation of human and rat HF samples from endocardial left ventricular homogenate. These results demonstrate the importance of increased RLC phosphorylation in the up-regulation of myocardial performance and suggest that reduced RLC phosphorylation is a key aspect of impaired contractile function in the diseased myocardium.National Institutes of Health, NHLBI Grant HL004232 12; Wellcome Trust Grants 091460/Z/10/Z, 092852/Z/10/Z, 092852/Z/10/Z; Biotechnology and Biological Sciences Research Council Grant BB/I019448/1; The British Heart Foundation

FRET characterisation for cross-bridge dynamics in single-skinned rigor muscle fibres

In this work we demonstrate for the first time the use of Förster resonance energy transfer (FRET) as an assay to monitor the dynamics of cross-bridge conformational changes directly in single muscle fibres. The advantage of FRET imaging is its ability to measure distances in the nanometre range, relevant for structural changes in actomyosin cross-bridges. To reach this goal we have used several FRET couples to investigate different locations in the actomyosin complex. We exchanged the native essential light chain of myosin with a recombinant essential light chain labelled with various thiol-reactive chromophores. The second fluorophore of the FRET couple was introduced by three approaches: labelling actin, labelling SH1 cysteine and binding an adenosine triphosphate (ATP) analogue. We characterise FRET in rigor cross-bridges: in this condition muscle fibres are well described by a single FRET population model which allows us to evaluate the true FRET efficiency for a single couple and the consequent donor–acceptor distance. The results obtained are in good agreement with the distances expected from crystallographic data. The FRET characterisation presented herein is essential before moving onto dynamic measurements, as the FRET efficiency differences to be detected in an active muscle fibre are on the order of 10–15% of the FRET efficiencies evaluated here. This means that, to obtain reliable results to monitor the dynamics of cross-bridge conformational changes, we had to fully characterise the system in a steady-state condition, demonstrating firstly the possibility to detect FRET and secondly the viability of the present approach to distinguish small FRET variations

Neural cytoskeleton capabilities for learning and memory

This paper proposes a physical model involving the key structures within the neural cytoskeleton as major players in molecular-level processing of information required for learning and memory storage. In particular, actin filaments and microtubules are macromolecules having highly charged surfaces that enable them to conduct electric signals. The biophysical properties of these filaments relevant to the conduction of ionic current include a condensation of counterions on the filament surface and a nonlinear complex physical structure conducive to the generation of modulated waves. Cytoskeletal filaments are often directly connected with both ionotropic and metabotropic types of membrane-embedded receptors, thereby linking synaptic inputs to intracellular functions. Possible roles for cable-like, conductive filaments in neurons include intracellular information processing, regulating developmental plasticity, and mediating transport. The cytoskeletal proteins form a complex network capable of emergent information processing, and they stand to intervene between inputs to and outputs from neurons. In this manner, the cytoskeletal matrix is proposed to work with neuronal membrane and its intrinsic components (e.g., ion channels, scaffolding proteins, and adaptor proteins), especially at sites of synaptic contacts and spines. An information processing model based on cytoskeletal networks is proposed that may underlie certain types of learning and memory