Localization, dynamics, and function of survivin revealed by expression of functional survivinDsRed fusion proteins in the living cell

Survivin, a member of the inhibitor of apoptosis protein family, has attracted growing attention due to its expression in various tumors and its potential application in tumor therapy. However, its subcellular localization and function have remained controversial: Recent studies revealed that survivin is localized at the mitotic spindle, binds caspases, and could thus protect cells from apoptosis. The cell cycle-dependent expression of survivin and its antiapoptotic function led to the hypothesis that survivin connects the cell cycle with apoptosis, thus providing a death switch for the termination of defective mitosis. In other studies, survivin was detected at kinetochores, cleavage furrow, and midbody, localizations being characteristic for chromosomal passenger proteins. These proteins are involved in cytokinesis as inferred from the observation that RNA interference and expression of mutant proteins led to cytokinesis defects without an increase in apoptosis. To remedy these discrepancies, we analyzed the localizations of a survivinDsRed fusion protein in HeLa cells by using confocal laser scanning microscopy and time-lapse video imaging. SurvivinDsRed was excluded from the interphase nucleus and was detected in centrosomes and at kinetochores. It dissociated from chromosomes at the anaphase/telophase transition and accumulated at the ends of polar microtubuli where it was immediately condensed to the midbody. Overexpression of both survivinDsRed and of a phosphorylation-defective mutant conferred resistance against apoptosis-inducing reagents, but only the overexpressed mutant protein caused an aberrant cytokinesis. These data characterize in detail the dynamics of survivin in vertebrate cells and confirm that survivin represents a chromosomal passenger protein.     

N-benzylideneaniline and N-benzylaniline are potent inhibitors of lignostilbene-alpha,beta-dioxygenase, a key enzyme in oxidative cleavage of the central double bond of lignostilbene

Lignostilbene-alpha,beta-dioxygenase (LSD, EC 1.13.11.43) is involved in oxidative cleavage of the central double bond of lignostilbene to form the corresponding aldehydes by a mechanism similar to those of 9-cis-epoxycarotenoid dioxygenase and beta-carotene 15,15'-dioxygenase, key enzymes in abscisic acid biosynthesis and vitamin A biosynthesis, respectively. In this study, several N-benzylideneanilines and amine were synthesized and examined for their efficacy as inhibitors of LSD. N-(4-Hydroxybenzylidene)-3-methoxyaniline was found to be a potent inhibitor with IC50 = 0.3 microM and N-(4-hydroxybenzyl)-3-methoxyaniline was also active with IC50 = 10 microM. The information obtained from the structure-activity relationships study here can aid in discovering inhibitors of both abscisic acid and vitamin A biosynthesis.     

Importance of Ca2+ influx by Na+/Ca2+ exchange under normal and sodium-loaded conditions in mammalian ventricles

Na⁺/Ca²⁺ exchange (NCX) is a major Ca²⁺ extrusion system in cardiac myocytes, but can also mediate Ca²⁺ influx and trigger sarcoplasmic reticulum Ca²⁺ release. Under conditions such as digitalis toxicity or ischemia/reperfusion, increased [Na⁺]_i may lead to a rise in [Ca²⁺]_i through NCX, causing Ca²⁺ overload and triggered arrhythmias. Here we used an agent which selectively blocks Ca²⁺ influx by NCX, KB-R7943 (KBR), and assessed twitch contractions and Ca²⁺ transients in rat and guinea pig ventricular myocytes loaded with indo-1. KBR (5 M) did not alter control steady-state twitch contractions or Ca²⁺ transients at 0.5 Hz in rat, but significantly decreased them in guinea pig myocytes. When cells were Na⁺-loaded by perfusion of strophanthidin (50 M), the addition of KBR reduced diastolic [Ca²⁺]_i and abolished spontaneous Ca²⁺ oscillations. In guinea pig papillary muscles exposed to substrate-free hypoxic medium for 60 min, KBR (10 M applied 10 min before and during reoxygenation) reduced both the incidence and duration of reoxygenation-induced arrhythmias. KBR also enhanced the recovery of developed tension after reoxygenation. It is concluded that (1) the importance of Ca²⁺ influx via NCX for normal excitation-contraction coupling is species-dependent, and (2) Ca²⁺ influx via NCX may be critical in causing myocardial Ca²⁺ overload and triggered activities induced by cardiac glycoside or reoxygenation.     

Identification and functional analysis of human Tom22 for protein import into mitochondria

Mitochondria have a receptor complex in the outer membrane which recognizes and translocates mitochondrial proteins synthesized in the cytosol. We report here the identification and functional analysis of human Tom22 (hTom22). hTom22 has an N-terminal negatively charged region exposed to the cytosol, a putative transmembrane region, and a C-terminal intermembrane space region with little negative charge. Tom22 forms a complex with Tom20, and its cytosolic domain functions as an import receptor as in fungi. An import inhibition assay, using pre-ornithine transcarbamylase (pOTC) derivatives and a series of hTom22 deletion mutants, showed that the C-terminal segment of the cytosolic domain is important for presequence binding, whereas the N-terminal domain is important for binding to the mature portion of pOTC. No evidence for pOTC interaction with the Tom22 intermembrane space domain was obtained. Binding studies revealed that the presequence is critical for pOTC binding to Tom20, whereas both the presequence and mature portion are important for binding to Tom22. A cell-free immunoprecipitation assay indicated that an internal segment of the Tom22 cytosolic domain is important for interaction with Tom20.     

Oligomeric tubulin in large transporting complex is transported via kinesin in squid giant axons

Slow axonal transport depends on an active mechanism that conveys cytosolic proteins. To investigate its molecular mechanism, we now constructed an in vitro experimental system for observation of tubulin transport, using squid giant axons. After injecting fluorescence-labeled tubulin into the axons, we monitored the movement of fluorescence by confocal laser scanning microscopy and fluorescence correlation spectroscopy. Here, from the pharmacological experiments and the functional blocking of kinesin motor protein by anti-kinesin antibody, we show that the directional movement of fluorescent profile was dependent on kinesin motor function. The fluorescent correlation function and estimated translational diffusion time revealed that tubulin molecule was transported in a unique form of large transporting complex distinct from those of stable polymers or other cytosolic protein.     

Effects of wortmannin on the kinetics of GVBD and the activities of the maturation-promoting factor and mitogen-activated protein kinase during bovine oocyte maturation in vitro

The present study was conducted with the objective of examining the effect of wortmanin, a specific PI 3-kinase inhibitor, on the kinetic of GVBD, and on the activities of the maturation-promoting factor (MPF) and mitogen-activated protein (MAP) kinase during bovine oocyte maturation. The time sequence for GVBD was not different between oocytes cultured with or without wortmannin. Most of the cultured oocytes were at the filamentous bivalents stage after 4 h of culture. Six hours after the start of culture, most of the oocytes possessed germinal vesicles with condensed bivalent, and by 10 h of culture nearly all of the cultured oocytes underwent GVBD. A gradual increase in MPF activity until 12 h of culture was observed in the presence and absence of wortmannin. A sharp decrease in MPF activity in oocytes cultured without wortmannin treatment was recorded at 14 h of culture. Thereafter, MPF regained activity, reaching a maximum level at 20 to 24 h of culture. For oocytes cultured with wortmannin, no decline in the activity of MPF was observed during the interval from 12 to 24 h of culture. For these oocytes the MPF activity remained nearly stable during this transition until the end of incubation. The presence of wortmannin in the maturation medium did not alter MAP kinase activity. Taken together, these observations indicate that inhibition of PI 3-kinase does not modulate the time sequence of GVBD or the pattern of MAP kinase activity in bovine oocytes. However, PI 3-kinase might be one of the molecules that regulate the sharp reduction in the activity of MPF during the MI/MII transition.     

Crystal structure of amylomaltase from thermus aquaticus, a glycosyltransferase catalysing the production of large cyclic glucans

Amylomaltase is involved in the metabolism of starch, one of the most important polysaccharides in nature. A unique feature of amylomaltase is its ability to catalyze the formation of cyclic amylose. In contrast to the well studied cyclodextrin glucanotransferases (CGTases), which synthesize cycloamylose with a ring size (degree of polymerization or DP) of 6-8, the amylomaltase from Thermus aquaticus produces cycloamyloses with a DP of 22 and higher. The crystal structure of amylomaltase from Thermus aquaticus was determined to 2.0 A resolution. It is a member of the alpha-amylase superfamily of enzymes, whose core structure consists of a (beta, alpha)(8) barrel. In amylomaltase, the 8-fold symmetry of this barrel is disrupted by several insertions between the barrel strands. The largest insertions are between the third and fifth barrel strands, where two insertions form subdomain B1, as well as between the second and third barrel strands, forming the alpha-helical subdomain B2. Whereas part of subdomain B1 is also present in other enzyme structures of the alpha-amylase superfamily, subdomain B2 is unique to amylomaltase. Remarkably, the C-terminal domain C, which is present in all related enzymes of the alpha-amylase family, is missing in amylomaltase. Amylomaltase shows a similar arrangement of the catalytic side-chains (two Asp residues and one Glu residue) as in previously characterized members of the alpha-amylase superfamily, indicating similar mechanisms of the glycosyl transfer reaction. In amylomaltase, a conserved loop of around eight amino acid residues is partially shielding the active center. This loop, which is well conserved among other amylomaltases, may sterically hinder the formation of small cyclic products.     

Visualization of the Dynamics of Synaptic Vesicle and Plasma Membrane Proteins in Living Axons

Newly synthesized membrane proteins are transported by fast axonal flow to their targets such as the plasma membrane and synaptic vesicles. However, their transporting vesicles have not yet been identified. We have successfully visualized the transporting vesicles of plasma membrane proteins, synaptic vesicle proteins, and the trans-Golgi network residual proteins in living axons at high resolution using laser scan microscopy of green fluorescent protein-tagged proteins after photobleaching. We found that all of these proteins are transported by tubulovesicular organelles of various sizes and shapes that circulate within axons from branch to branch and switch the direction of movement. These organelles are distinct from the endosomal compartments and constitute a new entity of membrane organelles that mediate the transport of newly synthesized proteins from the trans-Golgi network to the plasma membrane.