Blue-light-induced shift of the phototropic fluence-response curve in Pilobolus sporangiophores

The effects of preirradiation with blue light on the shift of the fluence-response curve for the first and the second positive curvatures were examined in Pilobolus crystallinus (Wiggers) Tode sporangiophores. A 1-min preirradiation with blue light at 47 or 960 nmol·m^-2 lowered the fluence-response curve for the first positive curvature and shifted the peak to a higher fluence. The fluence-response curve was shifted back to a lower fluence when a dark period was inserted between the preirradiation and the curvature-inducing light. This shift back to lower fluence was biphasic when the fluence was high (960 nmol · m^-2), indicating the participation of two components in the phototropic reaction for the first positive curvature.The fluence-response curve for the second positive curvature did not seem to be shifted to a higher fluence region when fluence was varied by varying exposure time. However, the fluence-response curve obtained by varying the fluence rate of a 20-min irradiation period indicated that the second positive curvature was also shifted to a higher-fluence region by preirradiation with blue light. A small shoulder appeared on the fluence-response curve when preirradiation at a high fluence rate was given.Abbreviations BL blue light - CIL curvature-inducing light     

Gradient-tailored excitation for single-quantum NMR spectroscopy of aqueous solutions

Summary A novel approach to tailored selective excitation for the measurement of NMR spectra in non-deuterated aqueous solutions (WATERGATE, WATER suppression by GraAdient-Tailored Excitation) is described. The gradient echo sequence, which effectively combines one selective 180° radiofrequency pulse and two field gradient pulses, achieves highly selective and effective water suppression. This technique is ideally suited for the rapid collection of multi-dimensional data since a single-scan acquisition produces a pure phase NMR spectrum with a perfectly flat baseline, at the highest possible sensitivity. Application to the fast measurement of 2D NOE data of a 2.2. mM solution of a double-stranded DNA fragment in 90% H₂O at 5 °C is presented.     

A two-dimensional nuclear Overhauser enhancement (2D NOE) experiment for the elucidation of complete proton-proton cross-relaxation networks in biological macromolecules

The recently developed technique of two-dimensional (2D) cross-relaxation spectroscopy is utilized for systematic measurements of selective nuclear Overhauser enhancements (NOE) in the high resolution ¹H nuclear magnetic resonance (NMR) spectra of biological macromolecules in solution. Compared to conventional one-dimensional NOE studies, the 2D NOE experiment has the principal advantage that it avoids detrimental effects arising from the limited selectivity of preirradiation in crowded spectral regions. Furthermore, it yields with a single instrument setting a complete network of NOE's between all the protons in the macromolecule. The resulting information on intramolecular proton-proton distances provides a new avenue for studies of the spatial structures of biopolymers.     

1-<Superscript>13</Superscript>C amino acid selective labeling in a <Superscript>2</Superscript>H<Superscript>15</Superscript>N background for NMR studies of large proteins

Isotope labeling by residue type (LBRT) has long been an important tool for resonance assignments at the limit where other approaches, such as triple-resonance experiments or NOESY methods do not succeed in yielding complete assignments. While LBRT has become less important for small proteins it can be the method of last resort for completing assignments of the most challenging protein systems. Here we present an approach where LBRT is achieved by adding protonated ¹⁴N amino acids that are ¹³C labeled at the carbonyl position to a medium for uniform deuteration and ¹⁵N labeling. This has three important benefits over conventional ¹⁵N LBRT in a deuterated back ground: (1) selective TROSY-HNCO cross peaks can be observed with high sensitivity for amino-acid pairs connected by the labeling, and the amide proton of the residue following the ¹³C labeled amino acid is very sharp since its alpha position is deuterated, (2) the ¹³C label at the carbonyl position is less prone to scrambling than the ¹⁵N at the α-amino position, and (3) the peaks for the 1-¹³C labeled amino acids can be identified easily from the large intensity reduction in the ¹H-¹⁵N TROSY-HSQC spectrum for some residues that do not significantly scramble nitrogens, such as alanine and tyrosine. This approach is cost effective and has been successfully applied to proteins larger than 40 kDa. Electronic Supplementary Material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Time efficient detection of protein–ligand interactions with the polarization optimized PO-WaterLOGSY NMR experiment

The identification of compounds that bind to a protein of interest is of central importance in contemporary drug research. For screening of compound libraries, NMR techniques are widely used, in particular the Water-Ligand Observed via Gradient SpectroscopY (WaterLOGSY) experiment. Here we present an optimized experiment, the polarization optimized WaterLOGSY (PO-WaterLOGSY). Based on a water flip-back strategy in conjunction with model calculations and numerical simulations, the PO-WaterLOGSY is optimized for water polarization recovery. Compared to a standard setup with the conventional WaterLOGSY, time consuming relaxation delays have been considerably shortened and can even be omitted through this approach. Furthermore, the robustness of the pulse sequence in an industrial setup was increased by the use of hard pulse trains for selective water excitation and water suppression. The PO-WaterLOGSY thus yields increased time efficiency by factor of 3–5 when compared with previously published schemes. These time savings have a substantial impact in drug discovery, since significantly larger compound libraries can be tested in screening campaigns. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Water–Protein Hydrogen Exchange in the Micro-Crystalline Protein Crh as Observed by Solid State NMR Spectroscopy

We report site-resolved observation of hydrogen exchange in the micro-crystalline protein Crh. Our approach is based on the use of proton T₂^′ -selective ¹H–¹³C–¹³C correlation spectra for site-specific assignments of carbons nearby labile protein protons. We compare the proton T₂^′ selective scheme to frequency selective water observation in deuterated proteins, and discuss the impacts of deuteration on ¹³C linewidths in Crh. We observe that in micro-crystalline proteins, solvent accessible hydroxyl and amino protons show comparable exchange rates with water protons as for proteins in solution, and that structural constraints, such as hydrogen bonding or solvent accessibility, more significantly reduce exchange rates.     

Biomass production of site selective 13C/15N nucleotides using wild type and a transketolase E. coli mutant for labeling RNA for high resolution NMR

Characterization of the structure and dynamics of nucleic acids by NMR benefits significantly from position specifically labeled nucleotides. Here an E. coli strain deficient in the transketolase gene (tktA) and grown on glucose that is labeled at different carbon sites is shown to facilitate cost-effective and large scale production of useful nucleotides. These nucleotides are site specifically labeled in C1′ and C5′ with minimal scrambling within the ribose ring. To demonstrate the utility of this labeling approach, the new site-specific labeled and the uniformly labeled nucleotides were used to synthesize a 36-nt RNA containing the catalytically essential domain 5 (D5) of the brown algae group II intron self-splicing ribozyme. The D5 RNA was used in binding and relaxation studies probed by NMR spectroscopy. Key nucleotides in the D5 RNA that are implicated in binding Mg²⁺ ions are well resolved. As a result, spectra obtained using selectively labeled nucleotides have higher signal-to-noise ratio compared to those obtained using uniformly labeled nucleotides. Thus, compared to the uniformly ¹³C/¹⁵N-labeled nucleotides, these specifically labeled nucleotides eliminate the extensive ¹³C–¹³C coupling within the nitrogenous base and ribose ring, give rise to less crowded and more resolved NMR spectra, and accurate relaxation rates without the need for constant-time or band-selective decoupled NMR experiments. These position selective labeled nucleotides should, therefore, find wide use in NMR analysis of biologically interesting RNA molecules.     

Changes in ionic selectivity with changes in density of water in gels and cells

Gels equilibrated with aqueous solutions of impermeant solutes reached a steady state in which, in the absence of a pressure difference, the activity of water in the pores of the gel was higher than that of water in the external solution. The chemical potential of water in the gel/polymer solution slurry was higher than that in the supernatant polymer solution removed from the gel. Water in the pores of the gel decreased in density to 0.96 as increasing osmotic stress was applied. It is argued that at constant temperature and pressure water can equilibrate between two compartments of unequal osmolality only by adjusting its molar volume. Experiments showed that when gel water had a higher activity than external water it was K⁺ selective; when it had a lower activity it was Na⁺ selective. It is proposed that a continuous spectrum of water structures can exist in these two compartment systems from dense, reactive, weakly-bonded water which selects highly hydrated ions, to expanded, stretched, unreactive, viscous water which is strongly hydrogen bonded and selects K⁺ and univalent anions. These findings are related to the state and properties of cytoplasmic water which is probably held under osmotic stress by the activity of the sodium pump.     

Mechanism of Prophylaxis by Silver Compounds against Infection of Burns

To clarify tthe mechanism by which local application of silver compounds protects burns against infection, an ion-specific electrode was used to measùre the concentration of silver ions in solutions. By this method it was shown that in burn dressings silver ions were reduced to a very low level by precipitation as silver chloride. The antibacterial effect was found to depend on the availability of silver ions from solution in contact with precipitate. Between 10^-5 and 10^-6 molar silver nitrate solution in water was rapidly bactericidal. The minimal amount of silver nitrate causing inhibition of respiration of skin in tissue culture was about 25 times the minimal concentration of silver nitrate that inhibited growth of Pseudomonas aeruginosa.     

N-terminal Proline-rich Domain Is Required for Scrambling Activity of Human Phospholipid Scramblases

Human phospholipid scramblase 1 (hPLSCR1), a type II integral class membrane protein, is known to mediate bidirectional scrambling of phospholipids in a Ca²⁺-dependent manner. hPLSCR2, a homolog of hPLSCR1 that lacks N-terminal proline-rich domain (PRD), did not show scramblase activity. We attribute this absence of scramblase activity of hPLSCR2 to the lack of N-terminal PRD. Hence to investigate the above hypothesis, we added the PRD of hPLSCR1 to hPLSCR2 (PRD-hPLSCR2) and checked whether scramblase activity was restored. Functional assays showed that the addition of PRD to hPLSCR2 restored scrambling activity, and deletion of PRD in hPLSCR1 (ΔPRD-hPLSCR1) resulted in a lack of activity. These results suggest that PRD is crucial for the function of the protein. The effects of the PRD deletion in hPLSCR1 and the addition of PRD to hPLSCR2 were characterized using various spectroscopic techniques. Our results clearly showed that hPLSCR1 and PRD-hPLSCR2 showed Ca²⁺-dependent aggregation and scrambling activity, whereas hPLSCR2 and ΔPRD-hPLSCR1 did not show aggregation and activity. Thus we conclude that scramblases exhibit Ca²⁺-dependent scrambling activity by aggregation of protein. Our results provide a possible mechanism for phospholipid scrambling mediated by PLSCRs and the importance of PRD in its function and cellular localization.     

Influence of erythrocyte shape on the rate of Ca2+-induced scrambling of phosphatidylserine

Membrane-perturbing agents that cause transformation of biconcave erythrocytes into echinocytes or stomatocytes were used to investigate the influence of erythrocyte shape on the rate of Ca²⁺-induced scrambling of phospholipids. Erythrocytes were treated with a variety of lipid-soluble compounds to induce these shape changes, followed by incubation with calcium and ionomycin to activate lipid scramblase. Prothrombinase activity of the cells was used to monitor the rate of surface exposure of phosphatidylserine, which is taken as a measure of scramblase activity. Echinocytes show an enhanced rate of scrambling, whereas stomatocytes show a reduced rate, relative to normocytes. This phenomenon appears to correlate with enhanced and diminished micro-exovesicle shedding from echinocytes and stomatocytes, respectively. It is concluded that the rate of calcium-induced phosphatidylserine exposure (rate of lipid scrambling) in erythrocytes depends for a considerable part on the cells' ability to form microvesicles.     

Transport of assimilates in the developing caryopsis of rice (Oryza sativa L.)

The movement of water in the dorsal region of the developing rice caryopsis was studied using solutions of the heavy metals lanthanum and uranium. In the electron microscope electron-opaque deposits were confined to the cell walls of the pigment strand indicating that this is the main route for the water which enters and leaves the caryopsis during grain filling. The pathway of assimilates into the developing caryopsis was examined using isolated caryopses which had taken up solutions of fluorescent dyes and also by autoradiography of caryopses which had transported ¹⁴C-labelled assimilates in vivo. The results show that assimilates unloaded from the phloem move through the pigment strand and circumferentially via cells of the nucellus before entering the endosperm. A scheme is presented for the interrelations of water and assimilate transport during grain filling     

Novel spectral-kinetic methods for investigation of ligand exchange in labile metal complexes in solutions

The luminescent spectral-kinetic method using selective complex excitation with short light pulse compared to relaxation reactions is described. The method makes it possible to obtain direct information on the rates of fast chemical reactions of dissociation or addition of ligands to Ln³⁺ ions in solutions. Data are presented on the rates and mechanisms of dissociation reactions for phenanthroline, bipyridile, salicylate, acetate, naphthoate and other complexes of Ln³⁺ ions in water and alcohols.     

Monitoring of membrane phospholipid scrambling in human erythrocytes and K562 cells with FM1-43 — a comparison with annexin V-FITC

The styryl dye FM1-43 becomes highly fluorescent upon binding to cell membranes. The breakdown of membrane phospholipid asymmetry in ionophore-stimulated T-lymphocytes further increases this fluorescence [Zweifach, 2000]. In this study, the capacity of FM1-43 to monitor membrane phospholipid scrambling was explored using flow cytometry in human erythrocytes and human erythrocyte progenitor K562 cells. The Ca²⁺-dependent phosphatidylserine-specific probe annexin V-FITC was used for comparison. The presented data show that the loss of phospholipid asymmetry that could be induced in human erythrocytes by elevated intracellular Ca²⁺ or by structurally different membrane intercalated amphiphilic compounds increases the FM1-43 fluorescence two- to fivefold. The profile of FM1-43 fluorescence for various treatments resembles that of phosphatidylserine exposure reported by annexin V-FITC. FM1-43 detected the onset of scrambling more efficiently than annexin V-FITC. The amphiphile-induced scrambling was shown to be a Ca²⁺-independent process. Monitoring of scrambling in K562 cells caused by NEM-induced Ca²⁺-release from intracellular stores and by Ca²⁺ and ionophore A23187 treatment showed that the increase in FM1-43 fluorescence correlated well with the number of annexin V-FITC-detected phosphatidylserine-positive cells. The results presented here show the usefulness of FM1-43 as a Ca²⁺-independent marker of dissipation in asymmetric membrane phospholipid distribution induced by various stimuli in both nucleated and non-nucleated cells.     

Efficient solvent suppression with adiabatic inversion for 1H-detected solid-state NMR

This study introduces a conceptually new solvent suppression scheme with adiabatic inversion pulses for ¹H-detected multidimensional solid-state NMR (SSNMR) of biomolecules and other systems, which is termed “Solvent suppression of Liquid signal with Adiabatic Pulse” (SLAP). ¹H-detected 2D ¹³C/¹H SSNMR data of uniformly ¹³C- and ¹⁵N-labeled GB1 sample using ultra-fast magic angle spinning at a spinning rate of 60 kHz demonstrated that the SLAP scheme showed up to 3.5-fold better solvent suppression performance over a traditional solvent-suppression scheme for SSNMR, MISSISSIPPI (Zhou and Rienstra, J Magn Reson 192:167–172, 2008) with 2/3 of the average RF power.

     

Water-protein NOEs: Optimized scheme for selective water excitation

An alternative scheme for selective water excitation is proposed. The pulse sequence saturates the resonances from the solute, allowing the observation of water-solute NOEs with low artifact levels. The water resonance is subsequently excited by a relatively non-selective 90° pulse. The scheme is compared to other selective water excitation schemes. 2D NOE-NOESY and ROE-NOESY pulse sequences are proposed which afford high sensitivity by efficient water excitation and flip-back by radiation damping, yet allow the use of short mixing times for the buildup of water-solute NOEs.     

Amino acid-selective isotope labeling of proteins for nuclear magnetic resonance study: Proteins secreted by Brevibacillus choshinensis

Here we report the first application of amino acid-type selective (AATS) isotope labeling of a recombinant protein secreted by Brevibacillus choshinensis for a nuclear magnetic resonance (NMR) study. To prepare the ¹⁵N-AATS-labeled protein, the transformed B. choshinensis was cultured in ¹⁵N-labeled amino acid-containing C.H.L. medium, which is commonly used in the Escherichia coli expression system. The analyses of the ¹H-¹⁵N heteronuclear single quantum coherence (HSQC) spectra of the secreted proteins with a ¹⁵N-labeled amino acid demonstrated that alanine, arginine, asparagine, cysteine, glutamine, histidine, lysine, methionine, and valine are suitable for selective labeling, although acidic and aromatic amino acids are not suitable. The ¹⁵N labeling for glycine, isoleucine, leucine, serine, and threonine resulted in scrambling to specific amino acids. These results indicate that the B. choshinensis expression system is an alternative tool for AATS labeling of recombinant proteins, especially secretory proteins, for NMR analyses.     

Disappearance rates of radiation-induced chromosome aberrations from swine leukocytes

Whole-blood leukocyte cultures were evaluated at intervals up to 20 weeks following the exposure of mature pigs to 150 or 200 R whole-body doses of γ-radiation. Lymphocyte number was depressed to approximately 35% of preirradiation values by 48 h postexposure; chromosome aberration levels also quite drastically during this period. A rapid decrease in aberrations per cell indicated selective removal of cells containing chromosome anomalues during the early postirradiation period. Elevated levels of both one-track and two-track aberrations persisted at 20 weeks although the former had been at preirradiation levels at 12 and 16 weeks.     

Studies on the Inhibition of Intestinal Absorption of Radioactive Strontium: III. The Effect of Administration of Sodium Alginate in Food and in Drinking Water

A method is reported which permits selective suppression of absorption of radioactive strontium from ingested food material, permitting calcium to be available to the body. Studies were carried out by measuring bone uptake of Sr⁸⁹ and Ca⁴⁵ when various amounts of sodium alginate were fed with the diet. Long-term studies were made in which two different levels of radioactivity were used, to determine the pattern of Sr⁸⁹ deposition with continuous intake of binding agent. It was found that administration of sodium alginate as a jelly overcomes the problem of constipation and effectively reduces Sr⁸⁹ uptake, up to 83%. This fact represents a significant finding with respect to the use of the compound in human subjects. Addition of sodium alginate to drinking water is effective with low levels of Sr⁸⁹ intake.This naturally occurring water-soluble macromolecular substance possesses several advantages in use for the suppression of absorption of radioactive strontium when compared with synthetic ion exchange resins: there is no disturbance of electrolyte balance; efficiency is not reduced by treatment over a prolonged period of time; and finally, the product is palatable.