Biodiversity of macrozoobenthos in a large river, the Austrian Danube, including quantitative studies in a free-flowing stretch below Vienna: a short review

The Danube is ca. 2850 km in length and is the second largest river in Europe. The Austrian part of the Danube falls 156 metres in altitude over its 351 km length and, since the early 1950s, the river has been developed into a power-generating waterway, so that the continuity of the river is now interrupted by ten impounded areas. Only two stretches of the original free-flowing river are left, the Wachau region (above river-km 2005, west of Vienna) and the region downstream from the impoundment at Vienna (river-km 1921). Most of the recent theories and concepts related to invertebrates, in the context of the ecology of running waters, are based on studies on small streams, whereas investigations of large rivers have played a minor role for a long time, mainly due to methodological difficulties. The authors' recent detailed studies on macroinvertebrates in the free-flowing section of the Danube below Vienna, provide an excellent opportunity to survey or restate scientific hypotheses on the basis of a large river. In this review the main interest focuses on the investigation of biodiversity, i.e. the number of species and their relative proportions in the whole invertebrate community, as well as major governing environmental factors. The article summarises the species composition, the important environmental variables at the river cross-section and the effect of upstream impoundment on the riverbed and its fauna.

DNA-mediated oxidation of transcription factor p53

Transcription factor p53 is the most commonly altered gene in human cancer. As a redox-active protein in direct contact with DNA, p53 can directly sense oxidative stress through DNA-mediated charge transport. Electron hole transport occurs with a shallow distance dependence over long distances through the π-stacked DNA bases, leading to the oxidation and dissociation of DNA-bound p53. The extent of p53 dissociation depends upon the redox potential of the response element DNA in direct contact with each p53 monomer. The DNA sequence dependence of p53 oxidative dissociation was examined by electrophoretic mobility shift assays using radiolabeled oligonucleotides containing both synthetic and human p53 response elements with an appended anthraquinone photooxidant. Greater p53 dissociation is observed from DNA sequences containing low redox potential purine regions, particularly guanine triplets, within the p53 response element. Using denaturing polyacrylamide gel electrophoresis of irradiated anthraquinone-modified DNA, the DNA damage sites, which correspond to locations of preferred electron hole localization, were determined. The resulting DNA damage preferentially localizes to guanine doublets and triplets within the response element. Oxidative DNA damage is inhibited in the presence of p53, however, only at DNA sites within the response element, and therefore in direct contact with p53. From these data, predictions about the sensitivity of human p53-binding sites to oxidative stress, as well as possible biological implications, have been made. On the basis of our data, the guanine pattern within the purine region of each p53-binding site determines the response of p53 to DNA-mediated oxidation, yielding for some sequences the oxidative dissociation of p53 from a distance and thereby providing another potential role for DNA charge transport chemistry within the cell.

To determine whether the change in p53 response element occupancy observed in vitro also correlates in cellulo, chromatin immunoprecipition (ChIP) and quantitative PCR (qPCR) were used to directly quantify p53 binding to certain response elements in HCT116N cells. The HCT116N cells containing a wild type p53 were treated with the photooxidant [Rh(phi)2bpy]³⁺, Nutlin-3 to upregulate p53, and subsequently irradiated to induce oxidative genomic stress. To covalently tether p53 interacting with DNA, the cells were fixed with disuccinimidyl glutarate and formaldehyde. The nuclei of the harvested cells were isolated, sonicated, and immunoprecipitated using magnetic beads conjugated with a monoclonal p53 antibody. The purified immounoprecipiated DNA was then quantified via qPCR and genomic sequencing. Overall, the ChIP results were significantly varied over ten experimental trials, but one trend is observed overall: greater variation of p53 occupancy is observed in response elements from which oxidative dissociation would be expected, while significantly less change in p53 occupancy occurs for response elements from which oxidative dissociation would not be anticipated.

The chemical oxidation of transcription factor p53 via DNA CT was also investigated with respect to the protein at the amino acid level. Transcription factor p53 plays a critical role in the cellular response to stress stimuli, which may be modulated through the redox modulation of conserved cysteine residues within the DNA-binding domain. Residues within p53 that enable oxidative dissociation are herein investigated. Of the 8 mutants studied by electrophoretic mobility shift assay (EMSA), only the C275S mutation significantly decreased the protein affinity (KD) for the Gadd45 response element. EMSA assays of p53 oxidative dissociation promoted by photoexcitation of anthraquinone-tethered Gadd45 oligonucleotides were used to determine the influence of p53 mutations on oxidative dissociation; mutation to C275S severely attenuates oxidative dissociation while C277S substantially attenuates dissociation. Differential thiol labeling was used to determine the oxidation states of cysteine residues within p53 after DNA-mediated oxidation. Reduced cysteines were iodoacetamide labeled, while oxidized cysteines participating in disulfide bonds were ¹³C₂D₂-iodoacetamide labeled. Intensities of respective iodoacetamide-modified peptide fragments were analyzed using a QTRAP 6500 LC-MS/MS system, quantified with Skyline, and directly compared. A distinct shift in peptide labeling toward ¹³C₂D₂-iodoacetamide labeled cysteines is observed in oxidized samples as compared to the respective controls. All of the observable cysteine residues trend toward the heavy label under conditions of DNA CT, indicating the formation of multiple disulfide bonds potentially among the C124, C135, C141, C182, C275, and C277. Based on these data it is proposed that disulfide formation involving C275 is critical for inducing oxidative dissociation of p53 from DNA.

Finite differences and a coupled analytic technique with applications to explosions and earthquakes

An analytic technique is developed that couples to finite difference calculations to extend the results to arbitrary distance. Finite differences and the analytic result, a boundary integral called two-dimensional Kirchhoff, are applied to simple models and three seismological problems dealing with data. The simple models include a thorough investigation of the seismologic effects of a deep continental basin. The first problem is explosions at Yucca Flat, in the Nevada test site. By modeling both near-field strong-motion records and teleseismic P-waves simultaneously, it is shown that scattered surface waves are responsible for teleseismic complexity. The second problem deals with explosions at Amchitka Island, Alaska. The near-field seismograms are investigated using a variety of complex structures and sources. The third problem involves regional seismograms of Imperial Valley, California earthquakes recorded at Pasadena, California. The data are shown to contain evidence of deterministic structure, but lack of more direct measurements of the structure and possible three-dimensional effects make two-dimensional modeling of these data difficult.

Quantitative, Time-Resolved Proteomic Analysis Using Bio-Orthogonal Non-Canonical Amino Acid Tagging

Bio-orthogonal non-canonical amino acid tagging (BONCAT) is an analytical method that allows the selective analysis of the subset of newly synthesized cellular proteins produced in response to a biological stimulus. In BONCAT, cells are treated with the non-canonical amino acid L-azidohomoalanine (Aha), which is utilized in protein synthesis in place of methionine by wild-type translational machinery. Nascent, Aha-labeled proteins are selectively ligated to affinity tags for enrichment and subsequently identified via mass spectrometry. The work presented in this thesis exhibits advancements in and applications of the BONCAT technology that establishes it as an effective tool for analyzing proteome dynamics with time-resolved precision.

Chapter 1 introduces the BONCAT method and serves as an outline for the thesis as a whole. I discuss motivations behind the methodological advancements in Chapter 2 and the biological applications in Chapters 2 and 3.

Chapter 2 presents methodological developments that make BONCAT a proteomic tool capable of, in addition to identifying newly synthesized proteins, accurately quantifying rates of protein synthesis. I demonstrate that this quantitative BONCAT approach can measure proteome-wide patterns of protein synthesis at time scales inaccessible to alternative techniques.

In Chapter 3, I use BONCAT to study the biological function of the small RNA regulator CyaR in Escherichia coli. I correctly identify previously known CyaR targets, and validate several new CyaR targets, expanding the functional roles of the sRNA regulator.

In Chapter 4, I use BONCAT to measure the proteomic profile of the quorum sensing bacterium Vibrio harveyi during the time-dependent transition from individual- to group-behaviors. My analysis reveals new quorum-sensing-regulated proteins with diverse functions, including transcription factors, chemotaxis proteins, transport proteins, and proteins involved in iron homeostasis.

Overall, this work describes how to use BONCAT to perform quantitative, time-resolved proteomic analysis and demonstrates that these measurements can be used to study a broad range of biological processes.

Quantitative data on the feeding of Cyclops strenuus Fischer and Cyclops viridis Jurine. [Translation from: Trudy saratov.Otd.vses.nauchno-issled.Inst.ozer.rech.ryb.Khoz. 1, 163-176, 1951.]

Cyclopids, exactly in the same way as daphnids, significant component in the nutrition of plankton-f and the young of the majority of fishes. It is established that the food spectrum of cyclopids is extremely broad: daphnids, planarians, Copepodite stages of copepods (cannibalism), rotifers, protists, bacteria, phytoplankton and so on. It is clear that the problem of studying these or other components of feeding in the general food spectrum can be definitely resolved only after obtaining exact quantitative data on the feeding of cyclopids. This article attempts to fill the gap in the study of the quantitative side of the feeding of cyclopias; in it is investigated the size of the 24-hour ration of cyclopids feeding on protists, the dependence of the ration on some factors of the external medium, and the difference of 24-hour consumption per unit weight of tody with two species of cyclopids (Cyclops strenuus and Cyclops viridis).

The changes in various iron compounds in culture media for algae. [Translation from: Archiv fur Hydrobiologie Supplemente Band 38(1/2) 151-169, 1970.]

The purpose of this work is a contribution to the quantitative record of the use of iron by planktonic algae. Preliminary experiments with Chlorella to determine the rate of iron intake in the presence of inorganic sources of iron did not produce the desired result. The crucial point of this work is the investigation of the influence of various external factors on the stability of FeEDTA (FeEDTA = Ferric(III)-compound of ethylene-diamine tetra-acetic acid), since this compound appears to be particularly well-suited as a source of iron for planktonic algae (e.g. TAMIYA et al. 1953). Cultures of Chlorella fusca in a light thermostat were used in experimental research. Methods and results are discussed.

Quantitative measurement of domain inversion in RuO2 : LiNbO3 crystal by digital holographic interferometry

We quantitatively study the domain inversion in a RuO2:LiNbO3 crystal wafer by the digital holographic interferometry. The crystal wafer is placed into one arm of a Mach-Zehnder-type interferometer to record a series of holograms. Making use of the angular spectrum backward propagation algorithm, we reconstruct the optical wave field in the crystal plane. The extracted phase difference from the reconstructed optical wave field is a well linear function of the applied external voltage. We deduce that the linear electro-optic coefficient of the detected RuO2:LiNbO3 crystal sample is 9.1x10(-12) m/V. An unexpected phase contrast at the antiparallel domain wall is observed and the influence of the applied external voltage on it is studied in detail. Also the built-in internal field is quantitatively measured as 0.72 kV/mm. (c) 2006 American Institute of Physics.

Water chemistry and osmoregulation in some arthropods, especially Malacostraca

This article briefly summarises some chief characteristics of osmoregulatory systems in malacostracan crustaceans, evolved to combat hydration, and the limitations thereby imposed on the salinity tolerance and distribution of these animals.

Design, Synthesis, and Study of Novel Platforms for Iron-N2 Chemistry and Photoinduced, Copper-mediated C-N Bond Formation

Several new ligand platforms designed to support iron dinitrogen chemistry have been developed. First, we report Fe complexes of a tris(phosphino)alkyl (CP^iPr₃) ligand featuring an axial carbon donor intended to conceptually model the interstitial carbide atom of the nitrogenase iron-molybdenum cofactor (FeMoco). It is established that in this scaffold, the iron center binds dinitrogen trans to the C_alkyl anchor in three structurally characterized oxidation states. Fe-C_alkyl lengthening is observed upon reduction, reflective of significant ionic character in the Fe-C_alkyl interaction. The anionic (CP^iPr₃)FeN₂^- species can be functionalized by a silyl electrophile to generate (CP^iPr₃)Fe-N₂SiR₃. This species also functions as a modest catalyst for the reduction of N₂ to NH₃. Next, we introduce a new binucleating ligand scaffold that supports an Fe(μ-SAr)Fe diiron subunit that coordinates dinitrogen (N₂-Fe(μ-SAr)Fe-N₂) across at least three oxidation states (Fe^IIFe^II, Fe^IIFe^I, and Fe^IFe^I). Despite the sulfur-rich coordination environment of iron in FeMoco, synthetic examples of transition metal model complexes that bind N₂ and also feature sulfur donor ligands remain scarce; these complexes thus represent an unusual series of low-valent diiron complexes featuring thiolate and dinitrogen ligands. The (N₂-Fe(μ-SAr)Fe-N₂) system undergoes reduction of the bound N₂ to produce NH₃ (~50% yield) and can efficiently catalyze the disproportionation of N₂H₄ to NH₃ and N₂. The present scaffold also supports dinitrogen binding concomitant with hydride as a co-ligand. Next, inspired by the importance of secondary-sphere interactions in many metalloenzymes, we present complexes of iron in two new ligand scaffolds ([SiP^NMe₃] and [SiP^iPr₂P^NMe]) that incorporate hydrogen-bond acceptors (tertiary amines) which engage in interactions with nitrogenous substrates bound to the iron center (NH₃ and N₂H₄). Cation binding is also facilitated in anionic Fe(0)-N₂ complexes. While Fe-N₂ complexes of a related ligand ([SiP^iPr₃]) lacking hydrogen-bond acceptors produce a substantial amount of ammonia when treated with acid and reductant, the presence of the pendant amines instead facilitates the formation of metal hydride species.

Additionally, we present the development and mechanistic study of copper-mediated and copper-catalyzed photoinduced C-N bond forming reactions. Irradiation of a copper-amido complex, ((m-tol)₃P)₂Cu(carbazolide), in the presence of aryl halides furnishes N-phenylcarbazole under mild conditions. The mechanism likely proceeds via single-electron transfer from an excited state of the copper complex to the aryl halide, generating an aryl radical. An array of experimental data are consistent with a radical intermediate, including a cyclization/stereochemical investigation and a reactivity study, providing the first substantial experimental support for the viability of a radical pathway for Ullmann C-N bond formation. The copper complex can also be used as a precatalyst for Ullmann C-N couplings. We also disclose further study of catalytic C_alkyl-N couplings using a CuI precatalyst, and discuss the likely role of [Cu(carbazolide)₂]^- and [Cu(carbazolide)₃]^- species as intermediates in these reactions.

Finally, we report a series of four-coordinate, pseudotetrahedral P₃Fe^II-X complexes supported by tris(phosphine)borate ([PhBP₃Fe^R]^-) and phosphiniminato X-type ligands (-N=PR'₃) that in combination tune the spin-crossover behavior of the system. Low-coordinate transition metal complexes such as these that undergo reversible spin-crossover remain rare, and the spin equilibria of these systems have been studied in detail by a suite of spectroscopic techniques.

Solar flare particle propagation--comparison of a new analytic solution with spacecraft measurements

A new analytic solution has been obtained to the complete Fokker-Planck equation for solar flare particle propagation including the effects of convection, energy-change, corotation, and diffusion with ĸ_r = constant and ĸ_Ɵ ∝ r². It is assumed that the particles are injected impulsively at a single point in space, and that a boundary exists beyond which the particles are free to escape. Several solar flare particle events have been observed with the Caltech Solar and Galactic Cosmic Ray Experiment aboard OGO-6. Detailed comparisons of the predictions of the new solution with these observations of 1-70 MeV protons show that the model adequately describes both the rise and decay times, indicating that ĸ_r = constant is a better description of conditions inside 1 AU than is ĸ_r ∝ r. With an outer boundary at 2.7 AU, a solar wind velocity of 400 km/sec, and a radial diffusion coefficient ĸ_r ≈ 2-8 x 10²⁰ cm²/sec, the model gives reasonable fits to the time-profile of 1-10 MeV protons from "classical" flare-associated events. It is not necessary to invoke a scatter-free region near the sun in order to reproduce the fast rise times observed for directly-connected events. The new solution also yields a time-evolution for the vector anisotropy which agrees well with previously reported observations.

In addition, the new solution predicts that, during the decay phase, a typical convex spectral feature initially at energy T_o will move to lower energies at an exponential rate given by T_KINK = T_oexp(-t/Ƭ_KINK). Assuming adiabatic deceleration and a boundary at 2.7 AU, the solution yields Ƭ_KINK ≈ 100h, which is faster than the measured ~200h time constant and slower than the adiabatic rate of ~78h at 1 AU. Two possible explanations are that the boundary is at ~5 AU or that some other energy-change process is operative.