Biomedical subjects
S S Andersen
Publications and source records attributed to S S Andersen.
Preparation of dissociated zebrafish spinal neuron cultures.
The method described here explains a simple protocol for how to prepare dissociated Zebrafish spinal neuron cultures. The neurons grow fast in a simple culture medium and at room temperature. Considering the advantages afforded by the optical transparency of the Zebrafish embryo combined with the powerful molecular perturbation techniques available, this technique has potential to further advance molecular analysis of axon growth and guidance.
Axon formation: a molecular model for the generation of neuronal polarity.
Neurons have unique structural and functional polarity. In general, information flows from the short dendrites to the long axon, and each neuron has multiple dendrites but only one axon. A detailed description of the cellular events leading to the establishment of axonal-dendritic polarity has been given from an in vitro hippocampal culture model system. Little is known, however, about the nature of the underlying molecular events. New data strongly suggest that actin depolymerization at a growth cone is crucial for axon fate determination. We hypothesize that an autocatalytic positive feedback loop at all growth cones locally regulates actin dynamics and other cellular events required for axon formation. Meanwhile, a negative feedback signal, produced by the positive feedback loop, propagates from all growth cones throughout the neuron and counteracts the positive feedback loops. Such feedback regulation provides a robust mechanism for spontaneous symmetry breaking and the formation of only one axon, even in a symmetric in vitro environment. Based on data from studies of cell migration, axon guidance, vesicle exocytosis, and the regulation of actin and microtubule polymerization, we propose a molecular scheme for the positive feedback loop and discuss possible negative feedback signals. BioEssays 22:172-179, 2000.
Spindle assembly and the art of regulating microtubule dynamics by MAPs and Stathmin/Op18.
The way that microtubules reorganize from their long, stable interphase configuration to form the mitotic spindle remains a challenging and unsolved question. It is now widely recognized that microtubule polymerization during the cell cycle is regulated by a balance between microtubule-stabilizing and-destabilizing factors. Stabilizing factors include a large group of microtubule-associated proteins (MAPs; e.g. MAP4, XMAP215, XMAP230/XMAP4 and XMAP310) and the destabilizing factors are a growing family of proteins (e.g. Stathmin/Op18 and XKCM1). Recent studies have allowed a mechanistic dissection of how these stabilizing and destabilizing factors regulate microtubule dynamics and spindle assembly.
Balanced regulation of microtubule dynamics during the cell cycle: a contemporary view.
Assembly of mitotic and meiotic spindles into an elliptical bipolar shape is an example of morphogenetic processes that involve local chromosomal regulation of microtubule dynamics for proper spatial microtubule assembly. Global microtubule dynamics during the cell cycle and local microtubule dynamics during spindle assembly are regulated by a balance between microtubule stabilizing and destabilizing factors. How a chromosome-induced phosphorylation gradient may be generated and modulate spindle microtubule assembly through balanced regulation of the activity of microtubule-associated proteins and Stathmin/Op 18 is analyzed.
Molecular characteristics of the centrosome.
As an organizer of the microtubule cytoskeleton in animals, the centrosome has an important function. From the early light microscopic observation of the centrosome to examination by electron microscopy, the centrosome field is now in an era of molecular identification and precise functional analyses. Tables compiling centrosomal proteins and reviews on the centrosome are presented here and demonstrate how active the field is. However, despite this intense research activity, many classical questions are still unanswered. These include those regarding the precise function of centrioles, the mechanism of centrosome duplication and assembly, the origin of the centrosome, and the regulation and mechanism of the centrosomal microtubule nucleation activity. Fortunately, these questions are becoming elucidated based on experimental data discussed here. Given the fact that the centrosome is primarily a site of microtubule nucleation, special focus is placed on the process of microtubule nucleation and on the regulation of centrosomal microtubule nucleation capacity during the cell cycle and in some tissues.
Genetic interactions between KAR7/SEC71, KAR8/JEM1, KAR5, and KAR2 during nuclear fusion in Saccharomyces cerevisiae.
During mating of Saccharomyces cerevisiae, two nuclei fuse to produce a single diploid nucleus. Two genes, KAR7 and KAR8, were previously identified by mutations that cause defects in nuclear membrane fusion. KAR7 is allelic to SEC71, a gene involved in protein translocation into the endoplasmic reticulum. Two other translocation mutants, sec63-1 and sec72Delta, also exhibited moderate karyogamy defects. Membranes from kar7/sec71Delta and sec72Delta, but not sec63-1, exhibited reduced membrane fusion in vitro, but only at elevated temperatures. Genetic interactions between kar7 and kar5 mutations were suggestive of protein-protein interactions. Moreover, in sec71 mutants, Kar5p was absent from the SPB and was not detected by Western blot or immunoprecipitation of pulse-labeled protein. KAR8 is allelic to JEMI, encoding an endoplasmic reticulum resident DnaJ protein required for nuclear fusion. Overexpression of KAR8/JEM1 (but not SEC63) strongly suppressed the mating defect of kar2-1, suggesting that Kar2p interacts with Kar8/Jem1p for nuclear fusion. Electron microscopy analysis of kar8 mutant zygotes revealed a nuclear fusion defect different from kar2, kar5, and kar7/sec71 mutants. Analysis of double mutants suggested that Kar5p acts before Kar8/Jem1p. We propose the existence of a nuclear envelope fusion chaperone complex in which Kar2p, Kar5p, and Kar8/Jem1p are key components and Sec71p and Sec72p play auxiliary roles.
Xenopus interphase and mitotic microtubule-associated proteins differentially suppress microtubule dynamics in vitro.
Based on observations of microtubule dynamics in Xenopus extracts and in vivo, it has been assumed that the pool of interphase microtubule-associated proteins (MAPs) are more potent microtubule stabilizers than their mitotic counterparts. The aim of this study was to test that assumption, and two questions were addressed here. First, are there differences in the composition of interphase and mitotic MAPs? Second, do interphase MAPs more potently promote microtubule assembly than mitotic MAPs? Biochemical purification from Xenopus egg extracts shows that the composition of interphase and mitotic MAPs is similar. XMAP215, XMAP230, and XMAP310, which are the three characterized Xenopus MAPs, show decreased microtubule binding in mitotic extracts, and mitotic MAPs are slightly more phosphorylated than interphase MAPs. Bulk polymerization and time-lapse video microscopy show that microtubules polymerized two times faster in the presence of total interphase MAPs compared with total mitotic MAPs. Interphase but not mitotic MAPs strongly promoted microtubule nucleation in solution. Video microscopy showed that microtubules never underwent catastrophes in the presence of either MAP fraction. It is proposed that the increase in microtubule dynamics at the onset of mitosis results from phosphorylation dependent decreased microtubule stabilization by MAPs, allowing destabilizing factors to increase the catastrophe frequency and dismantle the interphase microtubule network.
XMAP310: a Xenopus rescue-promoting factor localized to the mitotic spindle.
To understand the role of microtubule-associated proteins (MAPs) in the regulation of microtubule (MT) dynamics we have characterized MAPs prepared from Xenopus laevis eggs (Andersen, S.S.L., B. Buendia, J.E. Domínguez, A. Sawyer, and E. Karsenti. 1994. J. Cell Biol. 127:1289-1299). Here we report on the purification and characterization of a 310-kD MAP (XMAP310) that localizes to the nucleus in interphase and to mitotic spindle MTs in mitosis. XMAP310 is present in eggs, oocytes, a Xenopus tissue culture cell line, testis, and brain. We have purified XMAP310 to homogeneity from egg extracts. The purified protein cross-links pure MTs. Analysis of the effect of this protein on MT dynamics by time-lapse video microscopy has shown that it increases the rescue frequency 5-10-fold and decreases the shrinkage rate twofold. It has no effect on the growth rate or the catastrophe frequency. Microsequencing data suggest that XMAP230 and XMAP310 are novel MAPs. Although the three Xenopus MAPs characterized so far, XMAP215 (Vasquez, R.J., D.L. Gard, and L. Cassimeris. 1994. J. Cell Biol. 127:985-993), XMAP230, and XMAP310 are localized to the mitotic spindle, they have distinct effects on MT dynamics. While XMAP215 promotes rapid MT growth, XMAP230 decreases the catastrophe frequency and XMAP310 increases the rescue frequency. This may have important implications for the regulation of MT dynamics during spindle morphogenesis and chromosome segregation.
Mitotic chromatin regulates phosphorylation of Stathmin/Op18.
Meiotic and mitotic spindles are required for the even segregation of duplicated chromosomes to the two daughter cells. The mechanism of spindle assembly is not fully understood, but two have been proposed that are not mutually exclusive. The 'search and capture' model suggests that dynamic microtubules become progressively captured and stabilized by the kinetochores on chromosomes, leading to spindle assembly. The 'local stabilization' model proposes that chromosomes change the state of the cytoplasm around them, making it more favourable to microtubule polymerization. It has been shown that Stathmin/Op18 inhibits microtubule polymerization in vitro by interaction with tubulin, and that overexpression in tissue culture cells of non-phosphorylatable mutants of Stathmin/Op18 prevents the assembly of mitotic spindles. We have used Xenopus egg extracts and magnetic chromatin beads to show that mitotic chromatin induces phosphorylation of Stathmin/Op18. We have also shown that Stathmin/Op18 is one of the factors regulated by mitotic chromatin that governs preferential microtubule growth around chromosomes during spindle assembly.
The stathmin/tubulin interaction in vitro.
Stathmin is a highly conserved ubiquitous cytoplasmic protein, phosphorylated in response to extracellular signals and during the cell cycle. Stathmin has recently been shown to destabilize microtubules, but the molecular mechanisms of this function remained unclear. We show here that stathmin directly interacts with tubulin. We assessed the conditions of this interaction and determined some its quantitative parameters using plasmon resonance, gel filtration chromatography, and analytical ultracentrifugation. The stathmin/tubulin interaction leads to the formation of a 7.7 S complex with a 60-A Stokes radius, associating one stathmin with two tubulin heterodimer molecules as determined by direct quantification by Western blotting. This interaction is sensitive to pH and ionic environment. Its equilibrium dissociation constant, determined by plasmon resonance measurement of kinetic constants, has an optimum value of 0.5 microM at pH 6.5. The affinity was lowered with a fully "pseudophosphorylated" 4-Glu mutant form of stathmin, suggesting that it is modulated in vivo by stathmin phosphorylation. Finally, analysis of microtubule dynamics by video microscopy shows that, in our conditions, stathmin reduces the growth rate of microtubules with no effect on the catastrophe frequency. Overall, our results suggest that the stathmin destabilizing activity on microtubules is related to tubulin sequestration by stathmin.
Distinct roles of PP1 and PP2A-like phosphatases in control of microtubule dynamics during mitosis.
Assembly of a mitotic spindle requires the accurate regulation of microtubule dynamics which is accomplished, at least in part, by phosphorylation-dephosphorylation reactions. Here we have investigated the role of serine-threonine phosphatases in the control of microtubule dynamics using specific inhibitors in Xenopus egg extracts. Type 2A phosphatases are required to maintain the short steady-state length of microtubules in mitosis by regulating the level of microtubule catastrophes, in part by controlling the the microtubule-destabilizing activity and phosphorylation of Op18/stathmin. Type 1 phosphatases are only required for control of microtubule dynamics during the transitions into and out of mitosis. Thus, although both type 2A and type 1 phosphatases are involved in the regulation of microtubule dynamics, they have distinct, non-overlapping roles.
Purification of recombinant cyclin B1/cdc2 kinase from Xenopus egg extracts.
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Infections following epidural catheters.
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[Infections in connection with epidural catheterization].
Seventy-eight patients with culture-positive epidural catheters were studied. Fifty-nine had symptoms of exit site infection and 11 patients had clinical meningitis, two of whom also had en epidural abscess. This corresponds to a local infection incidence of at least 4.3% and an incidence of central nervous system infection of at least 0.7% at Odense University Hospital. The patients with generalized symptoms of infection had been catheterized for a longer time, and were older than patients with only local symptoms of infection. The microorganisms isolated from the epidural catheters were coagulase- negative staphylococci (41%), Staphylococcus aureus (35%), Gram-negative bacilli (14%) and other bacteria (10%). The Gram-negative bacilli and S. aureus caused serious infections more frequently than the others. We discuss the symptoms and diagnosis of spinal epidural abscess and propose prophylactic and diagnostic guidelines for epidural catheter-related infections.
[Ethylene glycol poisoning].
Ethylene glycol poisoning is a rare event in Denmark. The diagnosis can only be made as one of exclusion, since serum ethylene glycol measurements are not made in Danish laboratories. A case report of ethylene glycol poisoning is presented here. Diagnostic problems and treatment are described.
Infections following epidural catheterization.
Seventy-eight patients with culture-positive epidural catheters, were studied. Fifty-nine had symptoms of exit site infection and 11 patients had clinical meningitis, two of whom also had an epidural abscess. This corresponds to a local infection incidence of at least 4.3% and an incidence of central nervous system infection of at least 0.7% at Odense University Hospital. This degree of infection is of the same magnitude as that reported for intravascular devices. We found that the patients with generalized symptoms of infection had been catheterized for a longer time, and were older than patients with only local symptoms of infection. The microorganisms isolated from the tips of the epidural catheters were coagulase-negative staphylococci (41%), Staphylococcus aureus (35%), Gram-negative bacilli (14%) and others (10%). The Gram-negative bacilli and S. aureus caused serious infections more frequently than the others. We discuss the symptoms and diagnosis of spinal epidural abscess and suggest a proposal for prophylactic and diagnostic guidelines for epidural catheter-related infections.
[Poisoning with petroleum preparations--again!].
Accidentally occurring hydrocarbon poisoning during early childhood is a frequent and in most cases benign event. Nevertheless there is a potential risk of more serious consequences. We describe such a case. Respiratory symptoms are of the greatest concern. They include cough, tachypnoea, and in the more serious cases cyanosis and respiratory failure. Moreover, symptoms from the gastrointestinal tract are common. Treatment includes prevention of aspiration, symptomatic treatment and in more serious cases respiratory support.