Role and function of nonmuscle alpha-actinin-1 and -4 in regulating distinct subcategories of actin stress fibers in mammalian cells

Actin stress fibers are dynamic structures in the cytoskeleton, which respond to mechanical stimuli and affect cell motility, adhesion and invasion of cancer cells. In nonmuscle cells, stress fibers have been subcategorized to three distinct stress fiber types: dorsal and ventral stress fibers and transverse arcs. These stress fibers are dissimilar in their subcellular localization, connection to substratum as well as in their dynamics and assembly mechanisms. Still uncharacterized is how they differ in their function and molecular composition. Here, I have studied involvement of nonmuscle alpha-actinin-1 and -4 in regulating distinct stress fibers as well as their localization and function in human U2OS osteosarcoma cells. Except for the correlation of upregulation of alpha-actinin-4 in invasive cancer types very little is known about whether these two actinins are redundant or have specific roles. The availability of highly specific alpha-actinin-1 antibody generated in the lab, revealed localization of alpha-actinin-1 along all three categories of stress fibers while alphaactinin-4 was detected at cell edge, distal ends of stress fibers as well as perinuclear regions. Strikingly, by utilizing RNAi-mediated gene silencing of alpha-actinin-1 resulted in specific loss of dorsal stress fibers and relocalization of alpha-actinin-4 to remaining transverse arcs and ventral stress fibers. Unexpectedly, aberrant migration was not detected in cells lacking alpha-actinin-1 even though focal adhesions were significantly smaller and fewer. Whereas, silencing of alpha-actinin-4 noticeably affected overall cell migration. In summary, as part of my master thesis study I have been able to demonstrate distinct localization and functional patterns for both alpha-actinin-1 and -4. I have identified alpha-actinin-1 to be a selective dorsal stress fiber crosslinking protein as well as to be required for focal adhesion maturation, while alpha-actinin-4 was demonstrated to be fundamental for cell migration.

A particle filtering approach for structural system identification in vehicle–structure interaction problems

The problem of identification of parameters of a beam-moving oscillator system based on measurement of time histories of beam strains and displacements is considered. The governing equations of motion here have time varying coefficients. The parameters to be identified are however time invariant and consist of mass, stiffness and damping characteristics of the beam and oscillator subsystems. A strategy based on dynamic state estimation method, that employs particle filtering algorithms, is proposed to tackle the identification problem. The method can take into account measurement noise, guideway unevenness, spatially incomplete measurements, finite element models for supporting structure and moving vehicle, and imperfections in the formulation of the mathematical models. Numerical illustrations based on synthetic data on beam-oscillator system are presented to demonstrate the satisfactory performance of the proposed procedure.

Effect of mode of rolling on development of texture and microstructure in two-phase (alpha plus beta) brass

The evolution of microstructure and texture during deformation of two-phase (alpha + beta) brass was studied for different initial microstructure and texture. The deformation processing involved unidirectional and multi-step cross-rolling. The bulk textures were determined by measuring the pole figures and calculating the orientation distribution function ODF for both alpha (fcc) and beta (bcc) phases, while the microstructure and other microstructural parameters were measured through optical microscopy and scanning electron microscopy with electron back scatter diffraction (SEM-EBSD). Results indicate that textures developed after unidirectional rolling and multi-step cross-rolling are significantly different. The variation in initial texture had a pronounced effect on the development of texture in the alpha phase during subsequent deformation. (C) 2010 Elsevier B.V. All rights reserved.

Purification and characterization of an alpha-D-glucuronidase from a thermophilic fungus, Thermoascus aurantiacus

An alpha-D-glucuronidase was purified from the culture filtrates of Thermoascus aurantiacus. A simple colorimetric method for its assay is reported. The enzyme is a single polypeptide chain with a molecular weight of 118,000. It acts optimally at pH 4.5. It shows maximum activity at 65 degrees C. The t 1/2 at 70 degrees C was 40 min. It specifically cleaved the alpha-(1----2) linkage between 4-O-methyl-alpha-D-glucuronic acid and the xylose residue in xylan and several glucurono-xylooligosaccharides.

Increase in alpha-glycerophosphate dehydrogenase and other oxidoreductase activities of hepatic mitochondria on administration of vanadate to the rat

Liver mitochondria isolated from vanadate-administered rats showed increased (20-25%) rates of oxidation of both NAD(+)-linked substrates and succinate. Respiratory control index and ADP/O were unaffected by the treatment. Dormant and uncoupler-stimulated ATPase activity also was not affected by vanadate administration. Membrane-bound, electron-transport-linked dehydrogenase activities (both NAD(+)- and succinate-dependent) increased by 15-20% on vanadate treatment. Mitochondrial alpha-glycerophosphate dehydrogenase activity increased by 50% on vanadate administration. The above effects of vanadate on oxidoreductase activities could be prevented by the prior administration of antagonists to alpha-adrenergic receptors. Substrate-dependent H2O2 generation by mitochondria also showed an increase on vanadate administration.

Computer modelling approach to study the modes of binding of alpha- and beta-anomers of D-galactose, D-fucose and D-glucose to L-arabinose-binding protein

The modes of binding of alpha- and beta-anomers of D-galactose, D-fucose and D-glucose to L-arabinose-binding protein (ABP) have been studied by energy minimization using the low resolution (2.4 A) X-ray data of the protein. These studies suggest that these sugars preferentially bind in the alpha-form to ABP, unlike L-arabinose where both alpha- and beta-anomers bind almost equally. The best modes of binding of alpha- and beta-anomers of D-galactose and D-fucose differ slightly in the nature of the possible hydrogen bonds with the protein. The residues Arg 151 and Asn 232 of ABP from bidentate hydrogen bonds with both L-arabinose and D-galactose, but not with D-fucose or D-glucose. However in the case of L-arabinose, Arg 151 forms hydrogen bonds with the hydroxyl group at the C-4 atom and the ring oxygen, whereas in case of D-galactose it forms bonds with the hydroxyl groups at the C-4 and C-6 atoms of the pyranose ring. The calculated conformational energies also predict that D-galactose is a better inhibitor than D-fucose and D-glucose, in agreement with kinetic studies. The weak inhibitor D-glucose binds preferentially to one domain of ABP leading to the formation of a weaker complex. Thus these studies provide information about the most probable binding modes of these sugars and also provide a theoretical explanation for the observed differences in their binding affinities.

An alpha-aminomethyl carbanion equivalent via a novel Barbier reaction: (1H-naphtho[1,8-de]-1,2,3-triazin-2-yl)methyl anion

A novel sonication-promoted Barbier reaction putatively generated the titled species from the corresponding naphthotriazinylmethyl chloride and magnesium in THF: its formal addition to a variety of carbonyl compounds in situ occurred in excellent yields. Subsequent catalytic hydrogenolysis of the triazine moiety demasked the amine, thus defining a route to various phenylethylamines (including the alkaloid 'mescaline'), or ethanolamines (in two cases), in excellent overall yields. (C) 2000 Elsevier Science Ltd. All rights reserved.

Chiral synthons from carvone, Part 40. A simple, enantiospecific approach to both enantiomers of 1 alpha,25-dihydroxyvitamin D-3 A-ring precursors from R-carvone

A simple and direct approach to both enantiomeric series of A-ring derivatives of 1 alpha,25-dihydroxyvitamin D-3 and the corresponding 1 alpha,3 alpha-derivatives, starting from the abundantly available R-carvone, is described. (C) 2000 Elsevier Science Ltd. All rights reserved.

Conformations of peptides containing Z-alpha,beta-dehydroleucine (delta ZLeu). A comparison of Boc-Pro-delta ZLeu-Gly-NHEt and Boc-Pro-delta ZPhe-Gly-NHEt.

Two tripeptides of the type Boc-Pro-ΔZX-Gly-NHEt (where X = Leu, Phe) have been synthesized and their solution conformations investigated by 270 MHz 1H n.m.r. and i.r. spectroscopy. These conformational studies indicated that ΔZLeu, similar to ΔZPhe, has a strong tendency to stabilize folded Type II β-turn conformations when present at i + 2 position.

The hard-particle model for a dense granular flow down an inclined plane

Perfectly hard particles are those which experience an infinite repulsive force when they overlap, and no force when they do not overlap. In the hard-particle model, the only static state is the isostatic state where the forces between particles are statically determinate. In the flowing state, the interactions between particles are instantaneous because the time of contact approaches zero in the limit of infinite particle stiffness. Here, we discuss the development of a hard particle model for a realistic granular flow down an inclined plane, and examine its utility for predicting the salient features both qualitatively and quantitatively. We first discuss Discrete Element simulations, that even very dense flows of sand or glass beads with volume fraction between 0.5 and 0.58 are in the rapid flow regime, due to the very high particle stiffness. An important length scale in the shear flow of inelastic particles is the `conduction length' delta = (d/(1 - e(2))(1/2)), where d is the particle diameter and e is the coefficient of restitution. When the macroscopic scale h (height of the flowing layer) is larger than the conduction length, the rates of shear production and inelastic dissipation are nearly equal in the bulk of the flow, while the rate of conduction of energy is O((delta/h)(2)) smaller than the rate of dissipation of energy. Energy conduction is important in boundary layers of thickness delta at the top and bottom. The flow in the boundary layer at the top and bottom is examined using asymptotic analysis. We derive an exact relationship showing that the a boundary layer solution exists only if the volume fraction in the bulk decreases as the angle of inclination is increased. In the opposite case, where the volume fraction increases as the angle of inclination is increased, there is no boundary layer solution. The boundary layer theory also provides us with a way of understanding the cessation of flow when at a given angle of inclination when the height of the layer is decreased below a value h(stop), which is a function of the angle of inclination. There is dissipation of energy due to particle collisions in the flow as well as due to particle collisions with the base, and the fraction of energy dissipation in the base increases as the thickness decreases. When the shear production in the flow cannot compensate for the additional energy drawn out of the flow due to the wall collisions, the temperature decreases to zero and the flow stops. Scaling relations can be derived for h(stop) as a function of angle of inclination.

Activation barrier for the alpha.-cleavage process in thiones

The Occurrence of the Norrish type I a-cleavage process in some thio compounds has been examined by using the MIND013 method and employing the configuration interaction. Results reveal that where the radiationless process is not efficient, thio compounds can undergo photodissociation into radicals in their lowest triplet and singlet excited states. The activation barriers in all these cases arise from an avoided crossing between two states of different symmetries. The calculations of activation barriers by the CNDO-CI and MINDO-CI procedures reveal that the MINDO-CI method leads to realistic values of the activation energies.