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971.
Heiss M Fischer BE Jakob B Fournier C Becker G Taucher-Scholz G 《Radiation research》2006,165(2):231-239
The existing focusing heavy-ion microprobe at the Gesellschaft für Schwerionenforschung in Darmstadt (Germany) has been modified to enable the targeted irradiation of single, selected cells with a defined number of ions. With this setup, ions in the range from helium to uranium with linear energy transfers (LETs) up to approximately 15,000 keV/microm can be positioned with a precision of a few micrometers in the nuclei of single cells that are growing in culture on a thin polypropylene film. To achieve this accuracy, the microbeam traverses a thin vacuum window with minimal scattering. Electron emission from that window is used for particle detection. The cells are kept in a specially designed dish that is mounted directly behind the vacuum window in a setup allowing the precise movement and the imaging of the sample with microscopic methods. The cells are located by an integrated software program that also controls the rapid deflection and switching of the beam. In this paper, the setup is described in detail together with the first experiments showing its performance. We describe the ability of the microprobe to reliably hit randomly positioned etched nuclear tracks in CR-39 with single ions as well as the ability to visualize the ion hits using immunofluorescence staining for 53BP1 as a marker of DNA damage in the targeted cell nuclei. 相似文献
972.
Roman P. Jakob 《Journal of molecular biology》2009,387(4):1017-942
Prolyl cis/trans isomerizations determine the rates of many protein-folding reactions, and they can serve as molecular switches and timers. The energy required to shift the prolyl cis/trans equilibrium during these processes originates from conformational reactions that are linked structurally and energetically with prolyl isomerization. We used the N2 domain of the gene-3-protein of phage fd to elucidate how such an energetic linkage develops in the course of folding. The Asp160-Pro161 bond at the tip of a β hairpin of N2 is cis in the crystal structure, but in fact, it exists as a mixture of conformers in folded N2. During refolding, about 10 kJ mol− 1 of conformational energy becomes available for a 75-fold shift of the cis/trans equilibrium constant at Pro161, from 7/93 in the unfolded to 90/10 in the folded form. We combined single- and double-mixing kinetic experiments with a mutational analysis to identify the structural origin of this proline shift energy and to elucidate the molecular path for the transfer of this energy to Pro161. It originates largely, if not entirely, from the two-stranded β sheet at the base of the Pro161 hairpin. The two strands improve their stabilizing interactions when Pro161 is cis, and this stabilization is propagated to Pro161, because the connector peptides between the β strands and Pro161 are native-like folded when Pro161 is cis. In the presence of a trans-Pro161, the connector peptides are locally unfolded, and thus, Pro161 is structurally and energetically uncoupled from the β sheet. Such interrelations between local folding and prolyl isomerization and the potential modulation by prolyl isomerases might also be used to break and reestablish slow communication pathways in proteins. 相似文献
973.
Oxana V. Denisova Laura Kakkola Lin Feng Jakob Stenman Ashwini Nagaraj Johanna Lampe Bhagwan Yadav Tero Aittokallio Pasi Kaukinen Tero Ahola Suvi Kuivanen Olli Vapalahti Anu Kantele Janne Tynell Ilkka Julkunen Hannimari Kallio-Kokko Henrik Paavilainen Veijo Hukkanen Richard M. Elliott Jef K. De Brabander Xavier Saelens Denis E. Kainov 《The Journal of biological chemistry》2012,287(42):35324-35332
974.
Jakob Bunkenborg Guadalupe Espadas García Marcia Ivonne Peña Paz Jens S. Andersen Henrik Molina 《Proteomics》2010,10(16):3040-3044
Cell culture is a fundamental tool in proteomics where mammalian cells are cultured in vitro using a growth medium often supplemented with 5–15% FBS. Contamination by bovine proteins is difficult to avoid because of adherence to the plastic vessel and the cultured cells. We have generated peptides from bovine serum using four sample preparation methods and analyzed the peptides by high mass accuracy LC‐MS/MS. Distinguishing between bovine and human peptides is difficult because of a considerable overlap of identical tryptic peptide sequences. Pitfalls in interpretation, different database search strategies to minimize erroneous identifications and an augmented contaminant database are presented. 相似文献
975.
Alexander M. Walter Katrin Wiederhold Dieter Bruns Dirk Fasshauer Jakob B. S?rensen 《The Journal of cell biology》2010,188(3):401-413
Rapid neurotransmitter release depends on the ability to arrest the SNAP receptor (SNARE)–dependent exocytosis pathway at an intermediate “cocked” state, from which fusion can be triggered by Ca2+. It is not clear whether this state includes assembly of synaptobrevin (the vesicle membrane SNARE) to the syntaxin–SNAP-25 (target membrane SNAREs) acceptor complex or whether the reaction is arrested upstream of that step. In this study, by a combination of in vitro biophysical measurements and time-resolved exocytosis measurements in adrenal chromaffin cells, we find that mutations of the N-terminal interaction layers of the SNARE bundle inhibit assembly in vitro and vesicle priming in vivo without detectable changes in triggering speed or fusion pore properties. In contrast, mutations in the last C-terminal layer decrease triggering speed and fusion pore duration. Between the two domains, we identify a region exquisitely sensitive to mutation, possibly constituting a switch. Our data are consistent with a model in which the N terminus of the SNARE complex assembles during vesicle priming, followed by Ca2+-triggered C-terminal assembly and membrane fusion. 相似文献
976.
Barley thioredoxin h isozymes 1 (HvTrxh1) and barley thioredoxin h isozymes 2 (HvTrxh2) show distinct spatiotemporal distribution in germinating seeds. Using a novel approach involving measurement of bidirectional electron transfer rates between Escherichia coli thioredoxin, which exhibits redox-dependent fluorescence, and the barley isozymes, reaction kinetics and thermodynamic properties were readily determined. The reaction constants were ∼60% higher for HvTrxh1 than HvTrxh2, while their redox potentials were very similar. The primary nucleophile, CysN, of the active site Trp-CysN-Gly-Pro-CysC motif has an apparent pKa of 7.6 in both isozymes, as found by iodoacetamide titration, but showed ∼70% higher reactivity in HvTrxh1, suggesting significant functional difference between the isozymes. 相似文献
977.
Fernandes CG Borges CG Seminotti B Amaral AU Knebel LA Eichler P de Oliveira AB Leipnitz G Wajner M 《Cellular and molecular neurobiology》2011,31(5):775-785
Methylmalonic acidemia and propionic acidemia are organic acidemias biochemically characterized by predominant tissue accumulation
of methylmalonic acid (MMA) and propionic acid (PA), respectively. Affected patients present predominantly neurological symptoms,
whose pathogenesis is not yet fully established. In the present study we investigated the in vitro effects of MMA and PA on
important parameters of lipid and protein oxidative damage and on the production of reactive species in synaptosomes from
cerebrum of developing rats. Synaptosomes correspond to nerve terminals that have been used to investigate toxic properties
of compounds on neuronal cells. The in vivo effects of intrastriatal injection of MMA and PA on the same parameters and on
enzymatic antioxidant defenses, were also studied. MMA-induced in vitro and in vivo lipid peroxidation and protein oxidative
damage. Furthermore, the lipid oxidative damage was attenuated or prevented, pending on the doses utilized, by the free radical
scavengers α-tocopherol, melatonin and by the NMDA glutamate receptor antagonist MK-801, implying the involvement of reactive
species and glutamate receptor activation in these effects. In addition, 2′,7′-dichlorofluorescein diacetate oxidation was
significantly increased in synaptosomes by MMA, reinforcing that reactive species generation is elicited by this organic acid.
We also verified that glutathione peroxidase activity was inhibited by intrastriatal MMA injection. In contrast, PA did not
induce any significant effect on all parameters examined in vitro and in vivo, implying a selective action for MMA. The present
data demonstrate that oxidative stress is induced by MMA in vitro in nerve terminals and in vivo in striatum, suggesting the
participation of neuronal cells in MMA-elicited oxidative damage. 相似文献
978.
Sandberg Magdalena Widgren Bunkenborg Jakob Thyssen Stine Villadsen Martin Kofoed Thomas 《Amino acids》2022,54(4):601-613
Amino Acids - Granulocyte-macrophage colony-stimulating factor (GM-CSF) is a cytokine and a white blood cell growth factor that has found usage as a therapeutic protein. During analysis of... 相似文献
979.
Fernanda M. P. Oliveira Clarissa M. Knoechelmann Rainer Wirth Marcelo Tabarelli Inara R. Leal 《Biotropica》2023,55(2):444-453
Regenerating forests make up an increasingly large portion of tropical landscapes worldwide and regeneration dynamics may be influenced by leaf-cutting ants (LCA), which proliferate in disturbed areas and collect seeds for fungus culturing. Here, we investigate how LCA influences seed fate in human-modified areas of Caatinga dry forest. We evaluate the seed deposition and predation on Atta opaciceps nests, foraging habitat surrounding nest and control habitat away of nest influence of 15 colonies located along a forest cover gradient during the rainy and dry seasons. For each habitat, four 50-cm2 plots were established and all seeds on the soil surface were collected along 1 year. We recorded 13,628 seeds distributed among 47 species and 36.57% of the total seeds did not show any sign of predation. Nest mound habitats supported low-density and species-poor seed assemblages, which were taxonomically distinct from the control habitats. These effects only occurred in the rainy season. The proportion of undamaged seeds were similar across the habitats. While forest cover did not influence seed assemblage in terms of species richness or seed predation, it did interact with habitat type via increments in seed abundance as forest cover increased across the nests. Forest cover also affected seed composition, but only in the rainy season. These results indicate that LCA decrease seed deposition in areas under their influence, particularly on the nest mounds. As LCA profit from human disturbance in the Caatinga, their role as seed ‘sinks’ should be enhanced in disturbed Caatinga patches, particularly during the rainy season, when most of the plant recruitment occurs. Our findings reinforce the importance of LCA as drivers of forest dynamics and resilience in human-modified landscapes. 相似文献
980.
Christopher Mitchell Zachariah Wylde Enrique Del Castillo James Rapkin Clarissa M. House John Hunt 《Journal of evolutionary biology》2023,36(9):1266-1281
Although many theoretical models of male sexual trait evolution assume that sexual selection is countered by natural selection, direct empirical tests of this assumption are relatively uncommon. Cuticular hydrocarbons (CHCs) are known to play an important role not only in restricting evaporative water loss but also in sexual signalling in most terrestrial arthropods. Insects adjusting their CHC layer for optimal desiccation resistance is often thought to come at the expense of successful sexual attraction, suggesting that natural and sexual selection are in opposition for this trait. In this study, we sampled the CHCs of male black field crickets (Teleogryllus commodus) using solid-phase microextraction and then either measured their evaporative water loss or mating success. We then used multivariate selection analysis to quantify the strength and form of natural and sexual selection targeting male CHCs. Both natural and sexual selection imposed significant linear and stabilizing selection on male CHCs, although for very different combinations. Natural selection largely favoured an increase in the total abundance of CHCs, especially those with a longer chain length. In contrast, mating success peaked at a lower total abundance of CHCs and declined as CHC abundance increased. However, mating success did improve with an increase in a number of specific CHC components that also increased evaporative water loss. Importantly, this resulted in the combination of male CHCs favoured by natural selection and sexual selection being strongly opposing. Our findings suggest that the balance between natural and sexual selection is likely to play an important role in the evolution of male CHCs in T. commodus and may help explain why CHCs are so divergent across populations and species. 相似文献