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C Wiese

Publications and source records attributed to C Wiese.

At least 19 recordsLinked to original sources
PubMed2001

Different mechanisms of radiation-induced loss of heterozygosity in two human lymphoid cell lines from a single donor.

Allelic loss is an important mutational mechanism in human carcinogenesis. Loss of heterozygosity (LOH) at an autosomal locus is one outcome of the repair of DNA double-strand breaks (DSBs) and can occur by deletion or by mitotic recombination. We report that mitotic recombination between homologous chromosomes occurred in human lymphoid cells exposed to densely ionizing radiation. We used cells derived from the same donor that express either normal TP53 (TK6 cells) or homozygous mutant TP53 (WTK1 cells) to assess the influence of TP53 on radiation-induced mutagenesis. Expression of mutant TP53 (Met 237 Ile) was associated with a small increase in mutation frequencies at the hemizygous HPRT (hypoxanthine phosphoribosyl transferase) locus, but the mutation spectra were unaffected at this locus. In contrast, WTK1 cells (mutant TP53) were 30-fold more susceptible than TK6 cells (wild-type TP53) to radiation-induced mutagenesis at the TK1 (thymidine kinase) locus. Gene dosage analysis combined with microsatellite marker analysis showed that the increase in TK1 mutagenesis in WTK1 cells could be attributed, in part, to mitotic recombination. The microsatellite marker analysis over a 64-cM region on chromosome 17q indicated that the recombinational events could initiate at different positions between the TK1 locus and the centromere. Virtually all of the recombinational LOH events extended beyond the TK1 locus to the most telomeric marker. In general, longer LOH tracts were observed in mutants from WTK1 cells than in mutants from TK6 cells. Taken together, the results demonstrate that the incidence of radi-ation-induced mutations is dependent on the genetic background of the cell at risk, on the locus examined, and on the mechanisms for mutation available at the locus of interest.

Alleles↗
PubMed2001

Role of importin-beta in coupling Ran to downstream targets in microtubule assembly.

The guanosine triphosphatase Ran stimulates assembly of microtubule asters and spindles in mitotic Xenopus egg extracts. A carboxyl-terminal region of the nuclear-mitotic apparatus protein (NuMA), a nuclear protein required for organizing mitotic spindle poles, mimics Ran's ability to induce asters. This NuMA fragment also specifically interacted with the nuclear transport factor, importin-beta. We show that importin-beta is an inhibitor of microtubule aster assembly in Xenopus egg extracts and that Ran regulates the interaction between importin-beta and NuMA. Importin-beta therefore links NuMA to regulation by Ran. This suggests that similar mechanisms regulate nuclear import during interphase and spindle assembly during mitosis.

Animals↗
PubMed2001

Energy dissipation in photosynthesis: does the quenching of chlorophyll fluorescence originate from antenna complexes of photosystem II or from the reaction center?

Dissipation of light energy was studied in the moss Rhytidiadelphus squarrosus (Hedw.) Warnst., and in leaves of Spinacia oleracea L. and Arabidopsis thaliana (L.) Heynh., using chlorophyll fluorescence as an indicator reaction. Maximum chlorophyll fluorescence of 3-(3,4-dichlorophenyl)-1,1-dimethylurea (DCMU)-treated spinach leaves, as produced by saturating light and studied between and -20 degrees C, revealed an activation energy delta E of 0.11 eV. As this suggested recombination fluorescence produced by charge recombination between the oxidized primary donor of photosystem II and reduced pheophytin, a mathematical model explaining fluorescence, and based in part on known characteristics of primary electron-transport reactions, was developed. The model permitted analysis of different modes of fluorescence quenching, two localized in the reaction center of photosystem II and one in the light-harvesting system of the antenna complexes. It predicted differences in the relationship between quenching of variable fluorescence Fv and quenching of basal, so-called F0 fluorescence depending on whether quenching originated from antenna complexes or from reaction centers. Such differences were found experimentally, suggesting antenna quenching as the predominant mechanism of dissipation of light energy in the moss Rhytidiadelphus, whereas reaction-center quenching appeared to be important in spinach and Arabidopsis. Both reaction-center and antenna quenching required activation by thylakoid protonation but only antenna quenching depended on or was strongly enhanced by zeaxanthin. De-protonation permitted relaxation of this quenching with half-times below 1 min. More slowly reversible quenching, tentatively identified as so-called qI or photoinhibitory quenching, required protonation but persisted for prolonged times after de-protonation. It appeared to originate in reaction centers.

Bryopsida↗
PubMed2001

Ran stimulates spindle assembly by altering microtubule dynamics and the balance of motor activities.

The guanosine tri-phosphatase Ran stimulates assembly of microtubule spindles. However, it is not known what aspects of the microtubule cytoskeleton are subject to regulation by Ran in mitosis. Here we show that Ran-GTP stimulates microtubule assembly by increasing the rescue frequency of microtubules three- to eightfold. In addition to changing microtubule dynamics, Ran-GTP also alters the balance of motor activities, partly as a result of an increase in the amount of motile Eg5, a plus-end-directed microtubule motor that is essential for spindle formation. Thus, Ran regulates multiple processes that are involved in spindle assembly.

Animals↗
PubMed2001

Noninvasive mechanical ventilation improves endurance performance in patients with chronic respiratory failure due to thoracic restriction.

OBJECTIVE: Treatment with noninvasive mechanical ventilation (NMV) alleviates hypoventilation and improves gas exchange in patients with chronic respiratory failure (CRF) due to thoracic restriction. However, little is known about the effects of NMV on respiratory and peripheral muscle endurance. DESIGN: Prospective case-control study. SUBJECTS: Ten patients in clinically stable condition (age, 53.5 +/- 8.2 years [mean +/- SD]) with mild-to-moderate CRF due to thoracic restriction (PCO(2) between 45 mm Hg and 55 mm Hg) were treated with NMV during the night for 3 months. Ten matched patients (age, 52.2 +/- 9.5 years) receiving 3 months of normal care without NMV served as a control group. INTERVENTION AND MEASUREMENTS: After a 3-day period of familiarization with the endurance tests, all patients performed a baseline preintervention inspiratory threshold loading test, cycle ergometer test, and shuttle walking test on the same day. The endurance tests were then repeated following the 3-month intervention period. RESULTS: NMV was used on average for 7.1 +/- 0.9 h/d during the 3-month period. There was a significant improvement in endurance time (p < 0.0001) in all three endurance tests in the NMV group compared with the control group. In the NMV group, endurance time increased by 278 +/- 269% during the inspiratory threshold loading test, by 176 +/- 159% during the cycle ergometer test, and by 32 +/- 22% during the shuttle walking test. Significant improvements (p < 0.01) in both PO(2) and PCO(2) were also observed in the NMV group but not in the control group. CONCLUSIONS: Three months of treatment with NMV increases both respiratory and peripheral muscle endurance in patients with CRF due to thoracic restriction.

Case-Control Studies↗
PubMed2001

Mechanisms of mutagenesis in human cells exposed to 55 MeV protons.

Protons represent the major type of charged particle radiation in spaceflight environments. The purpose of this study was to assess mutations arising in human lymphoid cells exposed to protons. Mutations were quantitated at the thymidine kinase (TK1) locus in cell lines derived from the same donor: TK6 cells (wt TP53) and WTK1 cells (mutant TP53). WTK1 cells were much more susceptible to mutagenesis following proton exposure than TK6 cells. Intragenic deletions were observed among early-arising TK1 mutants in TK6 cells, but not in WTK1 cells where all of the mutants arose by LOH. Deletion was the predominant mode of LOH in TK6 cells, while allelic recombination was the major mode of LOH in WTK1 cells. Deletions were of variable lengths, from <1 cM to 64 cM, while mutations that arose by allelic recombination often extended to the telomere. In summary, proton exposures elicited many types of mutations at an autosomal locus in human cells. Most involved large scale loss of genetic information, either through deletion or by recombination.

Cell Line↗
PubMed2001

Genomic instability in human lymphoid cells exposed to 1 GeV/amu Fe ions.

The goal of this study was to assess whether charged particle radiations of importance to spaceflight elicit genomic instability in human TK6 lymphoblasts. The incidence of genomic instability in TK6 cells was assessed ~21 days after exposure to 2, 4, or 6 Fe ions (1 GeV/amu, LET= 146 keV/micrometers). Three indices of instability were used: intraclonal karyotypic heterogeneity, mutation rate analysis at the thymidine kinase (TK1) locus, and re-cloning efficiency. Fifteen of sixty clones demonstrated karyotypic heterogeneity. Five clones had multiple indicators of karyotypic change. One clone was markedly hypomutable and polyploid. Six clones were hypomutable, while 21 clones were mutators. Of these, seven were karyotypically unstable. Six clones had low re-cloning efficiencies, one of which was a mutator. All had normal karyotypes. In summary, many clones that survived exposure to a low fluence of Fe ions manifested one or more forms of genomic instability that may hasten the development of neoplasia through deletion or by recombination.

Cell Line↗
PubMed2000

Flexible coupling between light-dependent electron and vectorial proton transport in illuminated leaves of C3 plants. Role of photosystem I-dependent proton pumping.

The role of cyclic electron transport has been re-examined in leaves of C3 plants because the bioenergetics of chloroplasts (H +/e = 3 in the presence of a Q-cycle; H+/ATP = 4 of ATP synthesis) had suggested that cyclic electron flow has no function in C3 photosynthesis. After light activation of pea leaves, the dark reduction of P700 (the donor pigment of PSI) following far-red oxidation was much accelerated. This corresponded to loss of sensitivity of P700 to oxidation by farred light and a large increase in the number of electrons available to reduce P700+ in the dark. At low CO2 and O2 molar ratios, far-red light was capable of decreasing the activity of photosystem II (measured as the ratio of variable to maximal chlorophyll fluorescence, Fv/Fm) and of increasing light scattering at 535 nm and zeaxanthin synthesis, indicating formation of a trans-thylakoid pH gradient. Both the light-induced increase in the number of electrons capable of reducing far-redoxidised P700 and the decline in Fv/Fm brought about by far-red in leaves were prevented by methyl viologen. Antimycin A inhibited CO2-dependent O2 evolution of pea leaves at saturating but not under limiting light; in its presence, far-red light failed to decrease Fv/Fm. The results indicate that cyclic electron flow regulates the quantum yield of photosystem II by decreasing the intrathylakoid pH when there is a reduction in the availability of electron acceptors at the PSI level (e.g. during drought or cold stresses). It also provides ATP for the carbon-reduction cycle under high light. Under these conditions, the Q-cycle is not able to maintain a H+/e ratio of 3 for ATP synthesis: we suggest that the ratio is flexible, not obligatory.

Carbon Dioxide↗
PubMed2000

pH regulation in apoplastic and cytoplasmic cell compartments of leaves.

The regulation of pH in the apoplast, cytosol and chloroplasts of intact leaves was studied by means of fluorescent pH indicators and as a response of photosynthesis to acid stress. The apoplastic pH increased under anaerobiosis. Aeration reversed this effect. Apoplastic responses to CO2, HCl or NH3 differed considerably. Whereas HCl and ammonia caused rapid acidification or alkalinization, the return to initial pH values was slow after cessation of fumigation. Addition of CO2 either did not produce the acidification expected on the basis of known apoplastic buffering or even caused some alkalinization. Removal of CO2 shifted the apoplastic pH into the alkaline range before the pH returned to initial steady-state levels. In the presence of vanadate, the alkaline shift was absent and the apoplastic pH returned slowly to the initial level when CO2 was removed from the atmosphere. In contrast to the response of the apoplast, anaerobiosis acidified the cytosol or, in some species, had little effect on its pH. Acidification was rapidly reversed upon re-admission of oxygen. The CO2-dependent pH changes were very fast in the cytosol. Considerable alkalinization was observed after removal of CO2 under aerobic, but not under anaerobic conditions. Rates of the re-entry of protons into the cytosol during recovery from CO2 stress increased in the presence of oxygen with the length of previous exposure to high CO2. Effective pH regulation in the chloroplasts was indicated by the recovery of photosynthesis after the transient inhibition of photosynthetic electron flow when CO2 was increased from 0.038% to 16% in air. As photosynthesis became inhibited under high CO2, reduction of the electron transport chain increased transiently. The time required for recovery of photosynthesis from inhibition during persistent CO2 stress was similar to the time required for establishing steady-state pH values in the cytosol under acid stress. The high capacity of leaf cells for the rapid re-attainment of pH homeostasis in the apoplast and the cytoplasm under acid or alkaline stress suggested the rapid activation or deactivation of membrane-localised proton-transporting enzymes and corresponding ion channel regulation for co-transport of anions or counter-transport of cations together with proton fluxes. Acidification of the cytoplasm appeared to activate energy-dependent proton export primarily into the vacuoles whereas apoplastic alkalinization resulted in the pumping of protons into the apoplast. Proton export rates from the cytosol into the apoplast after anaerobiosis were about 100 nmol (m2 leaf area)(-1) s(-1) or less. Proton export under acid stress into the vacuole was about 1200 nmol m(-2) s(-1). The kinetics of pH responses to the addition or withdrawal of CO2 indicated the presence of carbonic anhydrase in the cytosol, but not in the apoplast.

Aerobiosis↗
PubMed2000

A new function for the gamma-tubulin ring complex as a microtubule minus-end cap.

Microtubule nucleation from centrosomes involves a lockwasher-shaped protein complex containing gamma-tubulin, named the gamma-tubulin ring complex (gammaTuRC). Here we investigate the mechanism by which the gammaTuRC nucleates microtubules, using a direct labelling method to visualize the behaviour of individual gammaTuRCs. A fluorescently-labelled version of the gammaTuRC binds to the minus ends of microtubules nucleated in vitro. Both gammaTuRC-mediated nucleation and binding of the gammaTuRC to preformed microtubules block further minus-end growth and prevent microtubule depolymerization. The gammaTuRC therefore acts as a minus-end-capping protein, as confirmed by electron-microscopic examination of gold-labelled gammaTuRCs. These data support a nucleation model for gammaTuRC function that involves capping of microtubules.

Animals↗
PubMed1999

Characterization of two related Drosophila gamma-tubulin complexes that differ in their ability to nucleate microtubules.

gamma-tubulin exists in two related complexes in Drosophila embryo extracts (Moritz, M., Y. Zheng, B.M. Alberts, and K. Oegema. 1998. J. Cell Biol. 142:1- 12). Here, we report the purification and characterization of both complexes that we name gamma-tubulin small complex (gammaTuSC; approximately 280,000 D) and Drosophila gammaTuRC ( approximately 2,200,000 D). In addition to gamma-tubulin, the gammaTuSC contains Dgrip84 and Dgrip91, two proteins homologous to the Spc97/98p protein family. The gammaTuSC is a structural subunit of the gammaTuRC, a larger complex containing about six additional polypeptides. Like the gammaTuRC isolated from Xenopus egg extracts (Zheng, Y., M.L. Wong, B. Alberts, and T. Mitchison. 1995. Nature. 378:578-583), the Drosophila gammaTuRC can nucleate microtubules in vitro and has an open ring structure with a diameter of 25 nm. Cryo-electron microscopy reveals a modular structure with approximately 13 radially arranged structural repeats. The gammaTuSC also nucleates microtubules, but much less efficiently than the gammaTuRC, suggesting that assembly into a larger complex enhances nucleating activity. Analysis of the nucleotide content of the gammaTuSC reveals that gamma-tubulin binds preferentially to GDP over GTP, rendering gamma-tubulin an unusual member of the tubulin superfamily.

Amino Acid Sequence↗
PubMed1999

Gamma-tubulin complexes and their interaction with microtubule-organizing centers.

Gamma-tubulin is as ubiquitous in eukaryotes as alpha- and beta-tubulin. Rather than forming part of the microtubule wall, however, gamma-tubulin is involved in microtubule nucleation. Although gamma-tubulin concentrates at microtubule-organizing centers, it also exists in a cytoplasmic complex whose size and complexity depends on the organism and cell type. In the past year, progress in understanding the functions of gamma-tubulin was made on two fronts: identifying the proteins that interact with gamma-tubulin and identifying the proteins that interact with the gamma-tubulin complex to tether it to the microtubule-organizing center.

Animals↗
PubMed1997

Myelin transcription factor 1 (Myt1) of the oligodendrocyte lineage, along with a closely related CCHC zinc finger, is expressed in developing neurons in the mammalian central nervous system.

The establishment and operation of the nervous system requires genetic regulation by a network of DNA-binding proteins, among which is the zinc finger superfamily of transcription factors. We have cloned and characterized a member of the unusual Cys-Cys-His-Cys (also referred to as Cys2HisCys, CCHC, or C2HC) class of zinc finger proteins in the developing nervous system. The novel gene, Myt1-like (Myt1l), is highly homologous to the original representative of this class, Myelin transcription factor 1 (Myt1) (Kim and Hudson, 1992). The MYT1 gene maps to human chromosome 20, while MYT1L maps to a region of human chromosome 2. Both zinc finger proteins are found in neurons at early stages of differentiation, with germinal zone cells displaying intense staining for MyT1. Unlike Myt1, Myt1l has not been detected in the glial lineage. Neurons that express Myt1l also express TuJ1, which marks neurons around the period of terminal mitosis. The Myt1l protein resides in distinct domains within the neuronal nucleus, analogous to the discrete pattern previously noted for Myt1 (Armstrong et al.: 14:303-321, 1995). The developmental expression and localization of these two multifingered CCHC proteins suggests that each may play a role in the development of neurons and oligodendroglia in the mammalian central nervous system.

Amino Acid Sequence↗
PubMed1997

Nuclear envelope assembly in Xenopus extracts visualized by scanning EM reveals a transport-dependent 'envelope smoothing' event.

We analyzed the pathway of nuclear envelope assembly in Xenopus egg extracts using field emission in-lens scanning electron microscopy. The binding, fusion, and flattening of vesicles onto the chromatin surface were visualized in detail. The first nuclear pore complexes assembled in flattened patches of nuclear envelope, before the chromatin was fully enclosed by membranes. Confirming previous transmission electron microscope observations, two morphologically distinct types of vesicles contributed to the nuclear membranes: ribosome-carrying ('rough') vesicles, many of which bound directly to chromatin, and 'smooth' vesicles, which appeared to associate primarily with other nuclear vesicles or membrane patches. The presence of ribosomes, an outer nuclear membrane marker, on many chromatin-binding vesicles suggested that chromatin-attachment proteins integral to the inner membrane were present on vesicles that also carried markers of the outer membrane and endoplasmic reticulum. Chromatin-associated vesicles also carried pore membrane proteins, since pore complexes formed when these vesicles were incubated with cytosol. A change in nuclear envelope morphology termed 'envelope smoothing' occurred 5-15 minutes after enclosure. Nuclear envelopes that were assembled in extracts depleted of wheat-germ-agglutinin-binding nucleoporins, and therefore unable to form functional pore complexes, remained wrinkled, suggesting that 'smoothing' required active nuclear transport. Lamins accumulated with time when nuclei were enclosed and had functional pore complexes, whereas lamins were not detected on nuclei that lacked functional pore complexes. Very low levels of lamins were detected on nuclear intermediates whose surfaces were substantially covered with patches of pore-complex-containing envelope, suggesting that pore complexes might be functional before enclosure.

Animals↗