The role of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (AcGEPC) and palmitoyl-lysophosphatidate in the responses of human blood platelets to collagen and thrombin

Desensitisation of human blood platelets to the effects of 1-O-alkyl-2-acetyl-sn-glyceryl-3-phosphorylcholine (1-O-alkylAcGEPC) and palmityl-lysophosphatidate by pre-incubation with these agonists has no effect on the aggregatory or secretory responses to collagen but causes 30–40% inhibition of these responses to thrombin in aspirin-treated platelets. The effects of 1-O-alkylAcGEPC and palmitoyl-lysophosphatidate are not additive. The results are not consistent with the proposal that 1-O-alkylAcGEPC or lysophosphatidate are the mediators for the responses to collagen observed when prostaglandinendo-peroxide synthesis is prevented, although they may play some role in the responses to thrombin under these conditions.     

Vimentin filaments and centrosomes: Are they associated?

HeLa cells were examined by immunofluorescence using anti-vimentin and anti-centrosphere anti-bodies, and by transmission electron microscopy (TEM), after vimentin redistribution induced by the action of nocodazole or taxol. A redistribution of vimentin bundles in the centriolar area was observed after nocodazole treatment, although no direct interaction between centrioles and vimentin filaments could be detected. After taxol treatment, the juxtanuclear accumulation of vimentin filaments and the centrioles were rarely observed in the same area. Our results do not support the concept of a direct association between centrioles and vimentin filaments.     

Effect of 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone on the secondary electron acceptor B of photosystem II

Measurements of chlorophyll fluorescence have been used to monitor electron transport from the primary electron acceptor of photosystem II, Q, to the secondary acceptor, B, in chloroplasts in either the presence or the absence of the plastoquinone analog 2,5-dibromo-3-methyl-6-isopropyl-p-benzoquinone (DBMIB). Electron transport is markedly slower from Q^? to either B or B^? in the presence of DBMIB. Binary oscillations in the rate of reoxidation of Q^? (equivalent to the reactions Q^?B → QB^? and Q^?B^? → QB^2?) after each of a series of flashes were of a phase opposite to those observed in the absence of DBMIB (J. M. Bowes, and A. R. Crofts, (1980) Biochim. Biophys. Acta590, 573–584). The results confirm that inhibition of electron transport by DBMIB in chloroplasts is not restricted to an inhibition of electron transfer from the plastoquinone pool, but that there is also a specific interaction between the reduced form of the inhibitor and the secondary electron acceptor B. Models are discussed to account for the mechanism of this interaction.     

Kinetics of the helix-coil transition of a polypeptide with non-ionic side groups, derived from ultrasonic relaxation measurements.

Ultrasonic absorption and velocity dispersion curves have been measured in the temperature induced helix-coil transition range of poly-N5-(3-hydroxypropyl)-L-glutamine in a methanol/water mixture. The results clearly reflect an effect due to the kinetics of the conformational conversion. A practically single relaxation time is observed which passes through a maximum when plotted versus the degree of transition. This maximum occurs at definitely less than 50% helix as predicted for by the theory for the comparatively short chain length involved here. The results are discussed in relation to previous theoretical and experimental findings.     

High performance liquid chromatography studies on the interactions of cis-Pt(NH3)2−(H2O)22+ with guanine and methylated g

Time dependence studies, using high performance liquid chromatography (HPLC), on the reaction between cis-diamminediaquoplatinum and guanine, N1-methylguanine, N7-methylguanine, N9-methylguanine, and N1 ,N7-dimethylguanine are reported. Each reaction gave rise to eight or more compounds; the major components have been prepared and characterization by ¹H and ¹⁹⁵Pt nuclear magnetic resonance has been attempted. Species of the form ((NH₃)₂Pt(NO₃)-(G-H)-(NO₃)-Pt(NH₃)₂)⁺, (NH₃)₂,Pt(G-H)(NO₃) monomer and (NH₃)₂Pt(G-H)(NO₃) dimer, where G-H indicates the guanine monoanion, are postulated.     

Regulation of gelsolin to plant actin filaments and its distribution in pollen

The dynamics of the actin cytoskeleton depends upon the unique constellation of ac- tin-binding proteins (ABPs), as well as their spatial distribution and local activation. However, the identification and characterization of actin-binding proteins in plant cells are still limited. At pre- sent, only a few plant ABPs have been identified in plant tissues, including profilin, ADF/cofilin, fimbrin, villin and several myosins. Compared with that in animals, there is still a long way for us …     

Prediction of the secondary and tertiary structure of plastocyanin.

The amino acid sequences of nine plastocyanins were examined using four published methods for the prediction of secondary structure in proteins. The results of the four methods were combined in such a way as to maximize agreement, and the position of alpha helices, beta sheets, and beta turns in plastocyanin was predicted. From this result and other information, such as the position of conserved residues and the requirements for coordination of copper, a preliminary model for the mainchain folding of the molecule was presented.     

Rate enhancement by guided diffusion. Chain length dependence of repressor-operator association rates

Association rates are calculated for cases where one reaction partner belongs to a chain that has an unspecific affinity to the other. Provided that the unspecific attachment does not completely suppress diffusion along the chain, this channeling may considerably speed up the association. Explicit formulae are derived to show how this effect depends on the chain length and other parameters. The influence of electrostatic forces and reaction barriers is discussed. Time dependent solutions of the diffusion equations are analyzed in order to test the usual steady state assumptions. Experiments on the repressor-operator system seem to be in good agreement with our theory.     

Uridine-cytidine kinase. Kinetic studies and reaction mechanism.

The initial velocity pattern has been determined for uridine-cytidine kinase purified from the murine mast cell neoplasm P815. With either uridine or cytidine as phosphate acceptor, and ATP as phosphate donor, the pattern observed was one of intersecting lines, ruling out a ping-pong reaction mechanism, and suggesting that the reaction probably proceeds by the sequential addition of both substrates to the enzyme to form a ternary complex, followed by the sequential release of the two products. This pattern was obtained whether the reaction was run in 0.01 m potassium phosphate buffer, pH 7.5, or in 0.1 m Tris-HCl, pH 7.2. When analyzed by the Sequen computer program, the data indicated an apparent K_m of the enzyme for uridine of 1.5 × 10^?4m, an apparent K_m for cytidine of 4.5 × 10^?5m, and a K_m for ATP, with uridine or cytidine as phosphate acceptor, of 3.6 × 10^?3m or 2.1 × 10^?3m, respectively. The V was 1.83 μmol phosphorylated/min/mg enzyme protein for the uridine kinase reaction and 0.91 μmol for the cytidine kinase reaction.     

Crystalline order to high resolution in the sheath of Methanospirillum hungatei: a cross-beta structure

Electron microscopy and electron diffraction indicate that the outer sheath of the cell wall of the archaebacterium Methanospirillum hungatei contains a two-dimensional crystalline lattice having, at least to low resolution, p2 symmetry in projection with a = 5.66 nm, b = 2.81 nm and gamma = 85.6 degrees. At a resolution of 2 nm, the unit cell contains two lobes, whereas high-angle electron diffraction shows the presence of a substantial quantity of beta structure, with the 0.47 nm spacing (between polypeptide chains within a sheet) oriented circumferentially. The sheath is unusual when compared to other regular surface arrays found on bacteria in that it is a compact structure with small subunits. It may have a structural role analogous to barrel hoops since it tends to fragment perpendicular to its axis to give rings or hoops.     

Letter: Myosin filaments showing a 430 A axial repeat periodicity

Synthetic myosin filaments with regular projections at intervals of 430 Å along their entire lengths have been observed using the electron microscope. The filaments were formed following dialysis in or rapid dilution to 0.3 m-KC1, 0.01 m-imidazole or phosphate buffer, pH 7.0. It is suggested that these filaments are made up of myosin molecules staggered by 430 Å.     

Endothelium-lymphocyte interactions in vitro. II. Adherence of allergized lymphocytes.

Peripheral blood lymphocytes from skin graft-sensitized pigs will adhere in vitro to fresh donor-type large vessel endothelium, but do not spread out or migrate. Similar cells will however spread out on and migrate through monolayers of cultured donor-type aortic endothelium to a significantly greater extent than nonallergized lymphocytes. Cells sensitized in mixed lymphocyte culture at first exhibit a nonspecific increase in adherence and migration correlated with increased thymidine uptake, but after more prolonged incubation adherence becomes specific for stimulator-type endothelium. It is suggested that lymphocyte infiltration of an allograft in the presence of circulating sensitized cells involves a combination of nonspecific lymphocyte adhesion to endothelium, antigenic stimulation of “primed” cells to increased motility, endothelial penetration and lymphokine production, and soluble-factor-mediated stimulation of migration by nonsensitized cells.     

Jasplakinolide reversibly disrupts actin filaments in suspension-cultured tobacco BY-2 cells

Summary. Jasplakinolide is potentially a useful pharmacological tool for the study of actin organization and dynamics in living cells, since it induces actin polymerization in vitro and, unlike phalloidin, is membrane permeative. In the present work, the effect of jasplakinolide on the actin cytoskeleton of living suspension-cultured Nicotiana tabacum ‘Bright Yellow 2’ cells was investigated. Actin filaments in the living cells were disrupted by jasplakinolide. The effect of jasplakionlide on the actin cytoskeleton was concentration and time dependent. When cells were treated with a moderate concentration (150 nM) of jasplakinolide, cortical actin filaments were disrupted preferentially, whereas actin aggregated at the perinuclear region. With concentrations higher than 400 nM and exposure times longer than 30 min, actin filaments in the cell disappeared completely. The effect of jasplakinolide on the actin cytoskeleton was reversible even at high concentration. Actin bundles appeared first in the perinuclear region within 5 min, and the cortical actin array was reestablished in 15 min, suggesting that actin filaments might be organized at this region. Received July 31, 2001 Accepted December 14, 2001     

Consecutive immobilized enzymatic reactions with and without enzyme denaturation

Consecutive biochemical reactions in an immobilized enzyme particle under the effects of internal and external diffusional resistances are analyzed. A rigorous nonlinear reaction kinetics is employed and the steady state effectiveness factor with negligible enzyme denaturation compared with the previous prediction by the first-order kinetics. It is found that the difference between them is rather substantial under most circumstances. The cases with significant enzyme denaturation are also investigated by using an unsteady state model. The substrate concentration responses to variation of the physical and kinetic parameters reveal many interesting characteristics of the reaction system.     

Regulation of insulin receptor kinase activity by insulin mimickers and an insulin antagonist

Three agents which mimic insulin action in intact cells (concanavalin A, wheat germ agglutinin, and polyclonal insulin receptor antibody), mimicked insulin's ability to stimulate the kinase activity of purified insulin receptors. In contrast, monoclonal insulin receptor antibody, an antagonist of insulin action, did not stimulate the phosphorylation of the insulin receptor either in intact IM-9 cells or in purified receptor preparations. This antibody, however, antagonized the ability of insulin to stimulate the phosphorylation of the receptor both in intact cells and in the purified receptor. These studies with insulin mimickers and an insulin antagonist are consistent with a role for the kinase activity of the receptor mediating the actions of insulin.     

The cytoskeletal framework of sea urchin eggs and embryos: developmental changes in the association of messenger RNA

Extraction of sea urchin eggs and embryos with Triton X-100 generated a cytoskeletal framework (CSK) composed of a cortical filamentous network and an internal system of filaments associated with ribosomes. The CSK contained only 10-20% of the cellular protein, RNA, and lipid. A specific subset of proteins was enriched in the CSK. Several lines of evidence suggest that mRNA is a component of the CSK of both eggs and embryos. First, the CSK contained poly(A) sequences which hybridized with [3H]poly(U). Second, the CSK contained polyribosomes. Finally, RNA extracted from the CSK showed translational activity in an in vitro system. The nonhistone messages present in the CSK were qualitatively similar to those solubilized by detergent, as determined by separation on polyacrylamide gels of the products of in vitro translation. In the unfertilized egg, most mRNA was present as nonpolyribosomal messenger ribonucleoprotein complexes which, along with monoribosomes, were efficiently extracted by Triton X-100. The converse was found in blastulae, as most of the mRNA was present as polyribosomes associated with the CSK, although monoribosomes were still efficiently extracted by detergent. These results indicate a correlation between the activation of protein synthesis in eggs and the association of polyribosomes with the CSK.