Rapid assignment of solution 31P NMR spectra of large proteins by solid-state spectroscopy

The application of the (31)P NMR spectroscopy to large proteins or protein complexes in solution is hampered by a relatively low intrinsic sensitivity coupled with large line widths. Therefore, the assignment of the phosphorus signals by two-dimensional NMR methods in solution is often extremely time consuming. In contrast, the quality of solid-state NMR spectra is not dependent on the molecular mass and the solubility of the protein. For the complex of Ras with the GTP-analogue GppCH(2)p we show solid-state (31)P NMR methods to be more sensitive by almost one order of magnitude than liquid-state NMR. Thus, solid-state NMR seems to be the method of choice for obtaining the resonance assignment of the phosphorus signals of protein complexes in solution. Experiments on Ras.GDP complexes show that the microcrystalline sample can be substituted by a precipitate of the sample and that unexpectedly the two structural states observed earlier in solution are present in crystals as well.     

On the seeding and oligomerization of pGlu-amyloid peptides (in vitro)

Oligomerization of amyloid beta (Abeta) peptides is the decisive event in the development of Alzheimer's disease (AD), the most common neurogenerative disorder in developed countries. Recent evidence links this conformation-driven process to primary- and secondary-structure modifications of Abeta. The N and C terminus of deposited Abeta has been shown to possess conspicuous heterogeneity. While the C-terminally longer form of Abeta, i.e., Abeta (42), is considered more amyloidogenic, the role of the N-terminal modifications, e.g., truncation and glutamate cyclization accounting for the majority of the deposited peptides, is less understood. In the present study, we characterized the oligomerization and seeding capacity of pGlu-amyloid peptides using two unrelated techniques based on flow cytometry or flourescence dye binding. Under different conditions and irrespective of the C terminus of Abeta, i.e., Abeta40 or 42, pGlu-modified peptides displayed an up to 250-fold accelerated initial formation of aggregates compared to unmodified Abeta. The accelerated seed formation is accompanied by a change in the oligomerization kinetics because of N-terminal pGlu formation. Furthermore, the formation of mixed aggregates consisting of either pGlu-Abeta (3-42) or ADan or ABri and Abeta (1-42) was investigated by Abeta fluorescence labeling in flow cytometry. The results suggest that pGlu-modified peptides are potential seeding species of aggregate formation in vivo. The data presented here and the abundance of pGlu peptides in amyloidoses, such as FBD and AD, suggest pGlu-amyloid peptides as a species with biophysical characteristics that might be in particular crucial for the initiation of the disease.     

Assembly and budding of Ebolavirus

Ebolavirus is responsible for highly lethal hemorrhagic fever. Like all viruses, it must reproduce its various components and assemble them in cells in order to reproduce infectious virions and perpetuate itself. To generate infectious Ebolavirus, a viral genome-protein complex called the nucleocapsid (NC) must be produced and transported to the cell surface, incorporated into virions, and then released from cells. To further our understanding of the Ebolavirus life cycle, we expressed the various viral proteins in mammalian cells and examined them ultrastructurally and biochemically. Expression of nucleoprotein alone led to the formation of helical tubes, which likely serve as a core for the NC. The matrix protein VP40 was found to be critical for transport of NCs to the cell surface and for the incorporation of NCs into virions, where interaction between nucleoprotein and the matrix protein VP40 is likely essential for these processes. Examination of virus-infected cells revealed that virions containing NCs mainly emerge horizontally from the cell surface, whereas empty virions mainly bud vertically, suggesting that horizontal budding is the major mode of Ebolavirus budding. These data form a foundation for the identification and development of potential antiviral agents to combat the devastating disease caused by this virus.     

Molecular determinants of Ebola virus virulence in mice

Zaire ebolavirus (ZEBOV) causes severe hemorrhagic fever in humans and nonhuman primates, with fatality rates in humans of up to 90%. The molecular basis for the extreme virulence of ZEBOV remains elusive. While adult mice resist ZEBOV infection, the Mayinga strain of the virus has been adapted to cause lethal infection in these animals. To understand the pathogenesis underlying the extreme virulence of Ebola virus (EBOV), here we identified the mutations responsible for the acquisition of the high virulence of the adapted Mayinga strain in mice, by using reverse genetics. We found that mutations in viral protein 24 and in the nucleoprotein were primarily responsible for the acquisition of high virulence. Moreover, the role of these proteins in virulence correlated with their ability to evade type I interferon-stimulated antiviral responses. These findings suggest a critical role for overcoming the interferon-induced antiviral state in the pathogenicity of EBOV and offer new insights into the pathogenesis of EBOV infection.     

Reverse Genetics Demonstrates that Proteolytic Processing of the Ebola Virus Glycoprotein Is Not Essential for Replication in Cell Culture

Ebola virus, a prime example of an emerging pathogen, causes fatal hemorrhagic fever in humans and in nonhuman primates. Identification of major determinants of Ebola virus pathogenicity has been hampered by the lack of effective strategies for experimental mutagenesis. Here we exploit a reverse genetics system that allows the generation of Ebola virus from cloned cDNA to engineer a mutant Ebola virus with an altered furin recognition motif in the glycoprotein (GP). When expressed in cells, the GP of the wild type, but not of the mutant, virus was cleaved into GP1 and GP2. Although posttranslational furin-mediated cleavage of GP was thought to be an essential step in Ebola virus infection, generation of a viable mutant Ebola virus lacking a furin recognition motif in the GP cleavage site demonstrates that GP cleavage is not essential for replication of Ebola virus in cell culture.     

Urea‐Modified Carbon Nitrides: Enhancing Photocatalytic Hydrogen Evolution by Rational Defect Engineering

The primary amine groups on the heptazine‐based polymer melon, also known as graphitic carbon nitride (g‐C₃N₄), can be replaced by urea groups using a two‐step postsynthetic functionalization. Under simulated sunlight and optimum Pt loading, this urea‐functionalized carbon nitride has one of the highest activities among organic and polymeric photocatalysts for hydrogen evolution with methanol as sacrificial donor, reaching an apparent quantum efficiency of 18% and nearly 30 times the hydrogen evolution rate compared to the nonfunctionalized counterpart. In the absence of Pt, the urea‐derivatized material evolves hydrogen at a rate over four times that of the nonfunctionalized one. Since “defects” are conventionally accepted to be the active sites in graphitic carbon nitride for photocatalysis, the work here is a demonstrated example of “defect engineering,” where the catalytically relevant defect is inserted rationally for improving the intrinsic, rather than extrinsic, photocatalytic performance. Furthermore, the work provides a retrodictive explanation for the general observation that g‐C₃N₄ prepared from urea performs better than those prepared from dicyandiamide and melamine. In‐depth analyses of the spent photocatalysts and computational modeling suggest that inserting the urea group causes a metal‐support interaction with the Pt cocatalyst, thus facilitating interfacial charge transfer to the hydrogen evolving centers.     

Differential responses of proliferative and non-proliferative leukemia cells to oxidative stress

The response of three human leukemia cell lines, the proliferative promonocyte THP-1 and the promyeloid HL60 cells and the non-proliferative phorbol ester-treated HL60 cells (HL60/PMA), to oxidative stress induced by tert-butylhydroperoxide (t-BHP) treatment was analyzed by fluorescence microplate assay, anti-oxidant enzyme activity measurements, high performance liquid chromatography, yopro-1/PI incorporation, poly (ADP-ribose) polymerase and caspase 3 cleavages. After t-BHP treatment, the non-proliferative HL60/PMA cells exhibited a weak increase in reactive oxygen species (ROS) production, a better preservation of thiol content, a decrease of glutathione peroxidase activity and a high ability to undergo necrosis rather than apoptosis. Submitted to the same treatment, the proliferative HL60 and THP-1 cells exhibited a high increase of ROS production, a moderate thiol depletion and a high percentage of apoptosis. Under thiol depleting conditions, the oxidative treatment of the HL60/PMA cells resulted in a high ROS production that reached levels similar to those of the two other cell lines and in cell death mainly by necrosis. In conclusion, these results that show proliferative phenotype is essential for cell response towards oxidative stress, are of particular interest in chemotherapy involving an oxidative mechanism.     

The influence of laser irradiation of low-power density on an experimental cartilage damage in rabbit knee-joints: an in vivo investigation considering macroscopic, histological and immunohistochemical changes]

In a total of 45 rabbits, knee-joint arthrosis was induced according to the Hulth & Telhag model. Depending on the post-operative survival time, the cartilage was investigated macroscopically, histologically and immunohistochemically (within a period of 10 days to 8 months). Thereafter, the influence of laser irradiation at a wavelength of 692.6 nm and energy densities of 1 and 4 J/cm2 on the cartilage morphology seven days following the exposure was examined. After joint instability surgery it was found out that the cartilage changes in the main stress area (MSA) and in regions outside the main stress area (ROMSA) progressed differently. Various qualitative and semi-quantitative changes were found for collagens I, II, IV and V, and for the glycoproteins fibronectin and tenascin. Immunohistochemically, there was a growing expression of collagen I in the apical layers, collagen II showed a stronger pericellular expression, and collagen IV showed, after an initial growth of the pericellular expression, a reduced territorial expression and a stronger apical-interterritorial expression in the osteoarthrotic cartilage. For fibronectin, the cellular expression turned out to grow in the ROMSA. In the MSA it decreased, but at the same time the interterritorial expression grew. For Tanascin, there was a decrease of the interterritorial expression in the radial zone while the pericellular and interterritorial expression of the apical layers of the osteoarthrotic cartilage grew. Lasing proved to significantly influence the osteoarthrotically changed cartilage when applied at an energy density of 1 J/cm2, i.e., the morphological changes had not yet progressed to the extent the control group had. Both the chondrocyte density and the glucosaminoglycan content turned out to be higher. When lasing was applied at higher energy densities, no significant difference among the control groups was found. Thus, it could be demonstrated in vivo that an arthrotic process decelerates through the influence of laser light of low-energy densities.     

Resolution of primary severe acute respiratory syndrome-associated coronavirus infection requires Stat1

Intranasal inhalation of the severe acute respiratory syndrome coronavirus (SARS CoV) in the immunocompetent mouse strain 129SvEv resulted in infection of conducting airway epithelial cells followed by rapid clearance of virus from the lungs and the development of self-limited bronchiolitis. Animals resistant to the effects of interferons by virtue of a deficiency in Stat1 demonstrated a markedly different course following intranasal inhalation of SARS CoV, one characterized by replication of virus in lungs and progressively worsening pulmonary disease with inflammation of small airways and alveoli and systemic spread of the virus to livers and spleens.     

Arabidopsis brassinosteroid-insensitive dwarf12 mutants are semidominant and defective in a glycogen synthase kinase 3beta-like kinase

Mutants defective in the biosynthesis or signaling of brassinosteroids (BRs), plant steroid hormones, display dwarfism. Loss-of-function mutants for the gene encoding the plasma membrane-located BR receptor BRI1 are resistant to exogenous application of BRs, and characterization of this protein has contributed significantly to the understanding of BR signaling. We have isolated two new BR-insensitive mutants (dwarf12-1D and dwf12-2D) after screening Arabidopsis ethyl methanesulfonate mutant populations. dwf12 mutants displayed the characteristic morphology of previously reported BR dwarfs including short stature, short round leaves, infertility, and abnormal de-etiolation. In addition, dwf12 mutants exhibited several unique phenotypes, including severe downward curling of the leaves. Genetic analysis indicates that the two mutations are semidominant in that heterozygous plants show a semidwarf phenotype whose height is intermediate between wild-type and homozygous mutant plants. Unlike BR biosynthetic mutants, dwf12 plants were not rescued by high doses of exogenously applied BRs. Like bri1 mutants, dwf12 plants accumulated castasterone and brassinolide, 43- and 15-fold higher, respectively, providing further evidence that DWF12 is a component of the BR signaling pathway that includes BRI1. Map-based cloning of the DWF12 gene revealed that DWF12 belongs to a member of the glycogen synthase kinase 3beta family. Unlike human glycogen synthase kinase 3beta, DWF12 lacks the conserved serine-9 residue in the auto-inhibitory N terminus. In addition, dwf12-1D and dwf12-2D encode changes in consecutive glutamate residues in a highly conserved TREE domain. Together with previous reports that both bin2 and ucu1 mutants contain mutations in this TREE domain, this provides evidence that the TREE domain is of critical importance for proper function of DWF12/BIN2/UCU1 in BR signal transduction pathways.