Colchicine activates actin polymerization by microtubule depolymerization

Dog exposure to doses from 39 to 124 Gy results in an increase of heart rate and tension in control of the cardiac rhythm, the extent being dose dependent. An additional physical load on the cardiovascular animals system reveals a high functional control reserves of the cardiovascular system which provides stable functioning even after exposure to the above doses.     

How (deficient) copper causes illness

Copper is an essential cofactor in all cells. However, it remains largely unknown how cells deal with this element, which is essential yet toxic. Through the study of microbial model systems on the one hand, and the investigation of inherited diseases in copper metabolism on the other, important insights into the way cells deal with copper can be gained. Two key new elements of copper metabolism have emerged from these studies: ATP-driven copper pumps and intracellular copper transport proteins, the copper chaperones.     

When overexpressed, a novel centrosomal protein, RanBPM, causes ectopic microtubule nucleation similar to gamma-tubulin

A novel human protein with a molecular mass of 55 kD, designated RanBPM, was isolated with the two-hybrid method using Ran as a bait. Mouse and hamster RanBPM possessed a polypeptide identical to the human one. Furthermore, Saccharomyces cerevisiae was found to have a gene, YGL227w, the COOH-terminal half of which is 30% identical to RanBPM. Anti-RanBPM antibodies revealed that RanBPM was localized within the centrosome throughout the cell cycle. Overexpression of RanBPM produced multiple spots which were colocalized with gamma-tubulin and acted as ectopic microtubule nucleation sites, resulting in a reorganization of microtubule network. RanBPM cosedimented with the centrosomal fractions by sucrose- density gradient centrifugation. The formation of microtubule asters was inhibited not only by anti- RanBPM antibodies, but also by nonhydrolyzable GTP-Ran. Indeed, RanBPM specifically interacted with GTP-Ran in two-hybrid assay. The central part of asters stained by anti-RanBPM antibodies or by the mAb to gamma-tubulin was faded by the addition of GTPgammaS-Ran, but not by the addition of anti-RanBPM anti- bodies. These results provide evidence that the Ran-binding protein, RanBPM, is involved in microtubule nucleation, thereby suggesting that Ran regulates the centrosome through RanBPM.     

PACAP-38 causes phospholipase C-dependent calcium signaling in rat acinar cell line

BACKGROUND: Pituitary adenylate cyclase activating peptide (PACAP-38), a neuropeptide of the vasoactive intestinal peptide/secretin family, localizes to intrapancreatic neurons and stimulates exocrine secretion from the pancreas. PACAP-38 stimulates calcium signaling in the rat pancreatic cell line AR42J. The purpose of this study was to elucidate the mechanisms of PACAP-evoked calcium signaling in these cells. METHODS: Continuous measurements of intracellular calcium were taken by fluorescent digital microscopy with the dye fura-2. Mechanisms of PACAP-38-evoked calcium signals were determined by a panel of inhibitors. Inositol phosphates production in response to PACAP-38 was measured. The ability of PACAP-38 to stimulate amylase release was used to determine a relevant dose range for these studies. RESULTS: We have shown that (1) AR42J cells respond to PACAP-38 with biphasic increases in [Ca2+]i in a dose-dependent fashion; (2) PACAP-38 acts through phospholipase C to release inositol triphosphate (IP3)-sensitive Ca2+ stores with (3) a subsequent influx of extracellular Ca2+. CONCLUSIONS: PACAP-38 activates calcium signaling through phospholipase C at concentrations that stimulate amylase release in AR42J cells.     

How to stabilize the level of ionized calcium and citrate during plateletpheresis

Felodipine is a calcium antagonist, one of the dihydropyridines, with potential application in transdermal therapeutic systems (TTS). Earlier studies reported that the high lag time of this drug limited its potential development in a TTS. The present study analyzes the effect of d-limonene at concentrations of 0.5, 1, 5 and 10% on the transdermal penetration of this drug. The study was performed using a diffusion technique in vitro, with the skin of the hairless rat. d-Limonene significantly reduced the lag time (Tl) to 1.4 h at a concentration of 1% (compared with 9.8 h in its absence). Higher concentrations did not produce a significant decrease in the value of this parameter. The presence of d-limonene in the formulae produces an increase in the permeability constant (Kp) and the flux (J). The relation between this increase and the percentage of d-limonene was non-linear. An asymptotic value was obtained at a concentration of 5%, with increases of 993% and 1570% for Kp and J, respectively.     

Tubulin-nucleotide interactions during the polymerization and depolymerization of microtubules

The interactions of nucleotides and their role in the polymerization of tubulin have been studied in detail. GTP promotes polymerization by binding to the exchangeable site (E site) of tubulin. The microtubules formed contain only GDP at the E site, indicating that hydrolysis of E site GTP occurs during or shortly after polymerization. Tubulin prepared by several cycles of polymerization and depolymerization will polymerize in the presence of ATP as well as GTP. Polymerization in ATP is preceded by a distinct lag period which is shorter at higher concentrations of ATP. As reported by others ATP will transphosphorylate bound GDP to GTP. Under polymerizing conditions the maximum level of GTP formation occurs at about the same time as the onset of polymerization, and the lag probably reflects the time necessary to transphosphorylate a critical concentration of tubulin. The transphosphorylated protein can be isolated and will polymerize without further addition of nucleotide. The transphosphorylated GTP is hydrolyzed and the phosphate released during polymerization. About 25% of the phosphate transferred from ATP is noncovalently bound to the subunit as inorganic phosphate and this fraction is also released during polymerization. The nonhydrolyzable analogue of GTP, GMPPNP, will promote microtubule assembly at high concentration. GMPPNP assembled microtubules do not depolymerize in Ca concentrations several fold greater than that which will completely depolymerize GTP assembled tubules; however, addition of Ca prior to inducing polymerization in GMPPNP prevents the formation of microtubules. Thus GTP hydrolysis appears to promote depolymerization rather than polymerization. GDP does not promote microtubule assembly but can inhibit GTP binding and GTP induced polymerization. GDP does not, however, induce the depolymerization of formed microtubules. These experiments demonstrate that tubulin polymerization can not be treated as a thermodynamically reversible process, but must involve one or more irreversible steps. Exchange experiments with [3H]GTP indicate that the "E" site on both microtubules and ring aggregates of tubulin is blocked and does not exchange rapidly. However, during polymerization and depolymerization induced by raising or lowering the temperature, respectively, all the E sites become transiently available and will exchange their nucleotide. This observation does not suggest a direct morphological transition between rings and microtubules. The presence of a blocked E site on the rings explains the apparent transphosphorylation and hydrolysis of "N" site nucleotide reported by others.     

Observation and quantification of individual microtubule behavior in vivo: microtubule dynamics are cell-type specific

Recent experiments have demonstrated that the behavior of the interphase microtubule array is cell-type specific: microtubules in epithelial cells are less dynamic than microtubules in fibroblasts (Pepper-kok et al., 1990; Wadsworth and McGrail, 1990). To determine which parameters of microtubule dynamic instability behavior are responsible for this difference, we have examined the behavior of individual microtubules in both cell types after injection with rhodamine-labeled tubulin subunits. Individual microtubules in both cell types were observed to grow, shorten, and pause, as expected. The average amount of time microtubules remained within the lamellae of CHO fibroblasts, measured from images acquired at 10-s intervals, was significantly shorter than the average amount of time microtubules remained within lamellae of PtK1 epithelial cells. Further analysis of individual microtubule behavior from images acquired at 2-s intervals reveals that microtubules in PtK1 cells undergo multiple brief episodes of growth and shortening, resulting in little overall change in the microtubule network. In contrast, microtubules in lamellae of CHO fibroblasts are observed to undergo fewer transitions which are of longer average duration, resulting in substantial changes in the microtubule network over time. A small subset of more stable microtubules was also detected in CHO fibroblasts. Quantification of the various parameters of dynamic instability behavior from these sequences demonstrates that the average rates of both growth and shortening are significantly greater for the majority of microtubules in fibroblasts than for microtubules in epithelial cells (19.8 +/- 10.8 microns/min, 32.2 +/- 17.7 microns/min, 11.9 +/- 6.5 microns/min, and 19.7 +/- 8.1 microns/min, respectively). The frequency of catastrophe events (1/interval between catastrophe events) was similar in both cell types, but the frequency of rescue events (1/time spent shrinking) was significantly higher in PtK1 cells. Thus, individual microtubules in PtK1 lamellae undergo frequent excursions of short duration and extent, whereas most microtubules in CHO lamellae undergo more extensive excursions often resulting in the appearance or disappearance of microtubules within the field of view. These observations provide the first direct demonstration of cell-type specific behavior of individual microtubules in living cells, and indicate that these differences can be brought about by modulation of the frequency of rescue. These results directly support the view that microtubule dynamic instability behavior is regulated in a cell-type specific manner.     

A quantitative analysis of microtubule elongation

Methods have been developed for differentially inhibiting microtubule nucleation and elongation in vitro. By use of polyanions, assembly-competent tubulin solutions of several milligrams/milliliter can be prepared which do not exhibit appreciable spontaneous assembly during the time-course of an experiment. Microtubule elongation can be initiated by the addition of known numbers of microtubule fragments. A detailed analysis of the resulting process demonstrates that: (a) rings are not obligatory intermediates in the nucleation sequence, and neither rings nor protofilament sheets are obligatory intermediates in the elongation reaction. (b) The end of an elongating microtubule often has a short region of open protofilament sheet or "C-microtubule" similar to that observed in vivo. (c) The development of turbidity follows a simple exponential approach to an equilibrium value. (d) The final equilibrium values are independent of the number of added nucleating fragments, while the initial growth rates and half-times to reach equilibrium are dependent on the number of added nuclei. (e) The final lengths of the microtubules at equilibrium are inversely proportional to the number of added fragments. (f) The equilibrium constants are independent of microtubule length. (g) The number of assembly and disassembly sites per microtubule is not a function of microtubule length. (h) The forward rate constants, the final polymer concentrations, and growth rates of microtubules are dependent upon the concentration of polyanion present. These results are strongly supportive of the idea that microtubule assembly is a "condensation-polymerization" and provide basic information on the kinetics and length distributions of the elongation in vitro.     

MAPs, MARKs and microtubule dynamics

Recent phylogenetic analyses reveal that many eukaryotic nuclear genes whose prokaryotic ancestry can be pinned down are of bacterial origin. Among them are genes whose products function exclusively in cytosolic metabolism. The results are surprising: we had come to believe that the eukaryotic nuclear genome shares a most recent common ancestor with archaeal genomes, thus most of its gene should be 'archaeal' (loosely speaking). Some genes of bacterial origin were expected as the result of transfer from mitochondria, of course, but these were thought to be relatively few, and limited to producing proteins reimported into mitochondria. Here, I suggest that the presence of many bacterial genes with many kinds of functions should not be a surprise. The operation of a gene transfer ratchet would inevitably result in the replacement of nuclear genes of early eukaryotes by genes from the bacteria taken by them as food.     

Glucocorticoid receptor inhibits microtubule assembly in vitro

The effect of glucocorticoid hormones, purified glucocorticoid receptor (GR) and purified heat shock protein M(r) 90,000 (hsp90) on microtubule (MT) assembly in vitro was tested by a spectrophotometric MT assembly assay and electron microscopy. GR significantly prolonged the nucleation phase, slowed down the assembly rate and reduced the maximal amplitude of MT assembly compared with control. The effects were partially reversed by the addition of glucocorticoid hormone. GR associated with MTs. These results indicate that GR affects MT assembly in vitro, which may be a functional correlate to the structural association of GR with MTs. This implies that factors affecting GR may affect MT assembly in vivo.     

Glycolytic enzymes and assembly of microtubule networks

The ability of glycolytic enzymes, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), aldolase, pyruvate kinase (PK), and lactate dehydrogenase muscle-type (LDH(M)), to generate interactive microtubule networks was investigated. Bundles have previously been defined as the parallel alignment of several microtubules and are one form of microtubule networks. Utilizing transmission electron microscopy, interactive networks of microtubules as well as bundles were readily observed in the presence of GAPDH, aldolase, or PK. These networks appear morphologically as cross-linked microtubules, oriented in many different ways. Light scattering indicated that the muscle forms of GAPDH, aldolase, PK and LDH(m) caused formation of the microtubule networks. Triose phosphate isomerase (TPI) and lactate dehydrogenase heart-type (LDH(H)), glycolytic enzymes which do not interact with tubulin or microtubules, did not produce bundles, or interactive networks. Sedimentation experiments confirmed that the enzymes that cross-link also co-pellet with the microtubules. Such cross-linking of microtubules indicate that the enzymes are multivalent with the capability of simultaneous binding to more than one microtubule.     

How prevalent is wishful thinking? Misattribution of arousal causes optimism and pessimism in subjective probabilities.

People appear to be unrealistically optimistic about their future prospects, as reflected by theory and research in the fields of psychology, organizational behavior, behavioral economics, and behavioral finance. Many real-world examples (e.g., consumer behavior during economic recessions), however, suggest that people are not always overly optimistic. I suggest that people can be both overly optimistic and pessimistic in their beliefs about future events, depending on whether they focus on success or on failure. More specifically, people judge the likelihood of desirable and undesirable events to be higher than similar neutral events because they misattribute the arousal those events evoke to their greater perceived likelihood. I demonstrated this stake-likelihood effect in 4 studies. In Study 1, arousal was shown to increase likelihood judgments. Study 2 demonstrated that such elevated likelihood judgments are due to misattribution of the arousal from having a stake in the outcome. Study 3 demonstrated that such misattribution of arousal occurs for desirable and undesirable events. Study 4 showed the effects of optimism and pessimism on likelihood judgments in a field setting with soccer fans. Together, the findings suggest that wishful thinking might be less prevalent than previously believed. Pessimism might be as likely as optimism in subjective probabilities. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Roles of nucleoside triphosphates in microtubule assembly

Depolymerization of microtubules in the ATP-reassembly buffer permitted the preparation of GDPETNGTP. Incubation of this tubulin fraction at 35 degrees with ATP induced the phosphorylation of E-site GDP into GTP, which was then dephosphorylated during microtubule assembly. Incubation of GDPETNGTP with phosphoenolpyruvate and pyruvate kinase [EC 2.7.1.40] also induced polymerization. Depolymerization of microtubules in the GTP-reassembly buffer yielded GTPETNGTP, which was capable of polymerizing into microtubules even in the absence of free GTP. In the presence of 4 M glycerol, GDPETNGTP assembled into microtubules with no change in the bound nucleotides.     

Antiproliferative mechanism of action of cryptophycin-52: kinetic stabilization of microtubule dynamics by high-affinity binding to microtubule ends

Cryptophycin-52 (LY355703) is a new synthetic member of the cryptophycin family of antimitotic antitumor agents that is currently undergoing clinical evaluation. At high concentrations (>/=10 times the IC50), cryptophycin-52 blocked HeLa cell proliferation at mitosis by depolymerizing spindle microtubules and disrupting chromosome organization. However, low concentrations of cryptophycin-52 inhibited cell proliferation at mitosis (IC50 = 11 pM) without significantly altering spindle microtubule mass or organization. Cryptophycin-52 appears to be the most potent suppressor of microtubule dynamics found thus far. It suppressed the dynamic instability behavior of individual microtubules in vitro (IC50 = 20 nM), reducing the rate and extent of shortening and growing without significantly reducing polymer mass or mean microtubule length. Using [3H]cryptophycin-52, we found that the compound bound to microtubule ends in vitro with high affinity (Kd, 47 nM, maximum of approximately 19.5 cryptophycin-52 molecules per microtubule). By analyzing the effects of cryptophycin-52 on dynamics in relation to its binding to microtubules, we determined that approximately 5-6 molecules of cryptophycin-52 bound to a microtubule were sufficient to decrease dynamicity by 50%. Cryptophycin-52 became concentrated in cells 730-fold, and the resulting intracellular cryptophycin-52 concentration was similar to that required to stabilize microtubule dynamics in vitro. The data suggest that cryptophycin-52 potently perturbs kinetic events at microtubule ends that are required for microtubule function during mitosis and that it acts by forming a reversible cryptophycin-52-tubulin stabilizing cap at microtubule ends.     

Inability to detect Chlamyodomonas microtubule assembly in vitro: possible implications to the in vivo regulation of microtubule assembly

It has been demonstrated that the in vitro assembly of microtubules from Chlamydomonas preparations does not occur under a wide range of conditions, including those efficacious for mammalian brain tubulin. This incompetence of Chlamydomonas extracts to form microtubules is independent of the tubulin concentration, the presence of added nucleotides or an added seed, temperature, or the concentration of divalent cation. However, an amorphous aggregate was observed under certain conditions, who composition was mainly tubulin. The in vitro reassembly of microtubules in gerbil brain extracts is inhibited by Chlamydomonas preparations. Fractionation of the Chlamydomonas extracts by column chromatography suggests that the inhibitory component is Chlamydomonas tubulin itself. The mechanism of this inhibition is unknown, but reassembly experiments indicate that the 2 types of tubulins cannot copolymerize. We suggest that the Chlamydomonas tubulin, derived from a cytoplasmic pool, requires to be activated prior to its in vivo polymerization into microtubules.     

alpha-crystallin stabilizes actin filaments and prevents cytochalasin-induced depolymerization in a phosphorylation-dependent manner

alpha-crystallin, a major lens protein of approximately 800 kDa with subunits of about 20 kDa has previously been shown to act as a chaperone protecting other proteins from stress-induced damage and to share sequence similarity with small heat-shock proteins, sHsp. It is now demonstrated that this chaperone effect extends to protection of the intracellular matrix component actin. It was found that the powerful depolymerization effect of cytochalasin D could be almost completely blocked by alpha-crystallin, alpha A-crystallin or alpha B-crystallin. However, phosphorylation of alpha-crystallin markedly decreased its protective effect. It is suggested that phosphorylation of alpha-crystallin may contribute to changes in actin structure observed during cellular remodeling that occurs with the terminal differentiation of a lens epithelial cell to a fiber cell and contributes to cellular remodeling in other cell types that contain alpha-crystallin species. This communication presents biochemical evidence clearly demonstrating that alpha-crystallin is involved in actin polymerization-depolymerization dynamics. It is also shown that alpha-crystallin prevented heat-induced aggregation of actin filaments. alpha-crystallin was found to stabilize actin polymers decreasing dilution-induced depolymerization rates up to twofold while slightly decreasing the critical concentration from 0.23 microM to 0.18 microM. Similar results were found with either alpha-crystallin or its purified subunits alpha A-crystallin and alpha B-crystallin. In contrast to the experiments with cytochalasin D, phosphorylation had no effect. There does not appear to be an interaction between alpha-crystallin and actin monomers since the effect of alpha-crystallin in enhancing actin polymerization does not become apparent until some polymerization has occurred. Examination of the stoichiometry of the alpha-crystallin effect indicates that 2-3 alpha-crystallin monomers/actin monomer give maximum actin polymer stabilization.