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

Regulatory and essential light chains of myosin rotate equally during contraction of skeletal muscle.

Myosin head consists of a globular catalytic domain and a long alpha-helical regulatory domain. The catalytic domain is responsible for binding to actin and for setting the stage for the main force-generating event, which is a "swing" of the regulatory domain. The proximal end of the regulatory domain contains the essential light chain 1 (LC1). This light chain can interact through the N and C termini with actin and myosin heavy chain. The interactions may inhibit the motion of the proximal end. In consequence the motion of the distal end (containing regulatory light chain, RLC) may be different from the motion of the proximal end. To test this possibility, the angular motion of LC1 and RLC was measured simultaneously during muscle contraction. Engineered LC1 and RLC were labeled with red and green fluorescent probes, respectively, and exchanged with native light chains of striated muscle. The confocal microscope was modified to measure the anisotropy from 0.3 microm(3) volume containing approximately 600 fluorescent cross-bridges. Static measurements revealed that the magnitude of the angular change associated with transition from rigor to relaxation was less than 5 degrees for both light chains. Cross-bridges were activated by a precise delivery of ATP from a caged precursor. The time course of the angular change consisted of a fast phase followed by a slow phase and was the same for both light chains. These results suggest that the interactions of LC1 do not inhibit the angular motion of the proximal end of the regulatory domain and that the whole domain rotates as a rigid body

Single Molecule Detection Approach to Muscle Study: Kinetics of a Single Cross-Bridge During Contraction of Muscle

D166V point mutation in the ventricular myosin regulatory light chain (RLC) is one of the causes of familial hypertrophic cardiomyopathy (FHC). We show here that the rates of cross-bridge attachment and dissociation are significantly different in isometrically contracting cardiac myofibrils from right ventricle of WT and Tg-D166V mice. To avoid averaging over ensembles of molecules composing muscle fibers, the data was collected from a single molecule. Kinetics were derived by tracking the orientation of a single actin molecule by fluorescence anisotropy. Orientation oscillated between two states, corresponding to the actin-bound and actin-free states of the myosin cross-bridge. The cross-bridge in a wild-type (healthy) heart stayed attached and detached from thin filament on average for 0.7 and 2.7 s, respectively. In FHC heart, these numbers increased to 2.5 and 5.8 s, respectively. These findings suggest that alterations in myosin cross-bridge kinetics associated with D166V mutation of RLC ultimately affect the ability of a heart to efficiently pump the blood

Enhancent fluorescent immunoassays on metal mirrors coated with metal particles

We present fluoroimmunoassays on plain metal coated surfaces (metal mirrors) enhanced by metal nanoparticles (silver island films, SIFs). Continuous metal layers (gold, or silver protected with a thin silica layer) were coated with SIFs. The surfaces were characterized by atomic force microscopy (AFM). The immunoassay (model assay for rabbit IgG, or myoglobin immunoassay) was performed on these surfaces using fluorescently labeled antibodies. Our results showed that SIFs alone (on glass surface not coated with metal) enhance the immunoassay signal approximately 3 to 10-fold. Using a thin metal layer deposited on glass as support for SIFs leads to up to 50-fold signal enhancement. We also present a theoretical explanation of localized nanoparticle polaritons