Inhibition of gliding movement by calcium in doublet microtubules on Tetrahymena ciliary dyneins in vitro

We examined the effects of Ca ions on the gliding movement of Tetrahymena ciliary doublet microtubules induced by 14S or 22S dyneins in an in vitro motility assay system. The doublet microtubule appeared as circular-arc in solution, about 5 to 6 μm in length [1]. The doublet microtubules glided distal-end first on a 14S or 22S dynein-coated glass surface either clockwise or counterclockwise following the addition of ATP. The diameter of the circular path changed according to Ca concentration in the solution. Gliding velocity was from 1 to 5 μm/s. The addition of 0.1% Nonidet P-4O was necessary to induce the gliding movement on 22S dynein. This movement on 22S dynein was strongly inhibited above 0.5 mM ATP in the presence of 10⁻⁹ M Ca, and at 0.05 to 1 mM ATP in the presence of 10⁻³ M Ca. Many studies have indicated that Ca ions regulate ciliary movement [2–8] in which dyneins and doublet microtubule in the axoneme may play an essential role. The inhibition of the gliding movement of doublet microtubule on dyneins at appropriate concentrations of Ca and ATP as observed in this study may be the key for understanding Ca regulation of ciliary motility.     

Studies on Oxalacetate Carboxylyase of Pig Heart Muscle

Oxalacetate carboxylyase was extracted from pig heart muscle and purified about 40 fold by means of acid and heat treatments, salting out and three steps of column chromatography. Some properties of the enzyme were studied by manometric and spectrophotometric measurements. The enzyme activity was optimal at pH 7. Km for oxalacetate was 4.3 × 10^?3 m/liter and the activation energy of the enzyme reaction was 15 kcal/m. The enzyme was activated by certain bivalent cations, among which Mn²⁺ was the most effective. Cu²⁺, Hg²⁺, some metal chelating reagents (EDTA, citrate and Oxalate) and pCMB strongly inhibited the enzyme activity. Inhibition by avidin was not observed, suggesting that biotin was not involved in the reaction as the prosthetic group of the enzyme.     

Particle Simulation of Oxidation Induced Band 3 Clustering in Human Erythrocytes

Oxidative stress mediated clustering of membrane protein band 3 plays an essential role in the clearance of damaged and aged red blood cells (RBCs) from the circulation. While a number of previous experimental studies have observed changes in band 3 distribution after oxidative treatment, the details of how these clusters are formed and how their properties change under different conditions have remained poorly understood. To address these issues, a framework that enables the simultaneous monitoring of the temporal and spatial changes following oxidation is needed. In this study, we established a novel simulation strategy that incorporates deterministic and stochastic reactions with particle reaction-diffusion processes, to model band 3 cluster formation at single molecule resolution. By integrating a kinetic model of RBC antioxidant metabolism with a model of band 3 diffusion, we developed a model that reproduces the time-dependent changes of glutathione and clustered band 3 levels, as well as band 3 distribution during oxidative treatment, observed in prior studies. We predicted that cluster formation is largely dependent on fast reverse reaction rates, strong affinity between clustering molecules, and irreversible hemichrome binding. We further predicted that under repeated oxidative perturbations, clusters tended to progressively grow and shift towards an irreversible state. Application of our model to simulate oxidation in RBCs with cytoskeletal deficiency also suggested that oxidation leads to more enhanced clustering compared to healthy RBCs. Taken together, our model enables the prediction of band 3 spatio-temporal profiles under various situations, thus providing valuable insights to potentially aid understanding mechanisms for removing senescent and premature RBCs.     

Laminin-based cell adhesion anchors microtubule plus ends to the epithelial cell basal cortex through LL5α/β

LL5β has been identified as a microtubule-anchoring factor that attaches EB1/CLIP-associating protein (CLASP)–bound microtubule plus ends to the cell cortex. In this study, we show that LL5β and its homologue LL5α (LL5s) colocalize with autocrine laminin-5 and its receptors, integrins α3β1 and α6β4, at the basal side of fully polarized epithelial sheets. Depletion of both laminin receptor integrins abolishes the cortical localization of LL5s, whereas LL5 depletion reduces the amount of integrin α3 at the basal cell cortex. Activation of integrin α3 is sufficient to initiate LL5 accumulation at the cell cortex. LL5s form a complex with the cytoplasmic tails of these integrins, but their interaction might be indirect. Analysis of the three-dimensional distribution of microtubule growth by visualizing EB1-GFP in epithelial sheets in combination with RNA interference reveals that LL5s are required to maintain the density of growing microtubules selectively at the basal cortex. These findings reveal that signaling from laminin–integrin associations attaches microtubule plus ends to the epithelial basal cell cortex.     

Arabidopsis HDA6 is required for freezing tolerance

The control of energy homeostasis within the hypothalamus is under the regulated control of homeostatic hormones, nutrients and the expression of neuropeptides that alter feeding behavior. Elevated levels of palmitate, a predominant saturated fatty acid in diet and fatty acid biosynthesis, alter cellular function. For instance, a key mechanism involved in the development of insulin resistance is lipotoxicity, through increased circulating saturated fatty acids. Although many studies have begun to determine the underlying mechanisms of lipotoxicity in peripheral tissues, little is known about the effects of excess lipids in the brain. To determine these mechanisms we used an immortalized, clonal, hypothalamic cell line, mHypoE-44, to demonstrate that palmitate directly alters the expression of molecular clock components, by increasing Bmal1 and Clock, or by decreasing Per2, and Rev-erbα, their mRNA levels and altering their rhythmic period within individual neurons. We found that these neurons endogenously express the orexigenic neuropeptides NPY and AgRP, thus we determined that palmitate administration alters the mRNA expression of these neuropeptides as well. Palmitate treatment causes a significant increase in NPY mRNA levels and significantly alters the phase of rhythmic expression. We explored the link between AMPK and the expression of neuropeptide Y using the AMPK inhibitor compound C and the AMP analog AICAR. AMPK inhibition decreased NPY mRNA. AICAR also elevated basal NPY, but prevented the palmitate-mediated increase in NPY mRNA levels. We postulate that this palmitate-mediated increase in NPY and AgRP synthesis may initiate a detrimental positive feedback loop leading to increased energy consumption.     

12′-Hydroxyl group remarkably reduces Roridin E cytotoxicity

Roridin E is a well-known macrocyclic trichothecene mycotoxin possessing potent antiproliferative activity against cancer cell lines. 12′-Hydroxyroridin E was isolated from a marine-derived fungus, Myrothecium roridum 98F42. The cytotoxicities of these two compounds were tested against human monocytic THP-1, human promyelocytic leukemia HL-60, and Chinese hamster V79 cells, and roridin E exhibited more than 1000-fold stronger cytotoxicity than its 12′-OH derivative; therefore, it was suggested that the 12′-position is closely involved in the cytotoxicity of these compounds.