Effect of pendant group structure on the hydrolytic stability of polyaspartamide polymers under physiological conditions

We describe the synthesis of metal chelating polymers based on polyaspartamide and polyglutamide backbones as carriers for (111)In in radioimmunoconjugates. These polymers [PAsp(DTPA), PGlu(DTPA)] have a biotin end group and diethylenetriaminepentaacetic acid (DTPA) chelators attached to the primary amines of the diethylenetriamine (DET) pendant groups of biotin-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} [PAsp(DET)] and of biotin-poly{N'-[N-(2-aminoethyl)-2-aminoethyl]glutamide} [PGlu(DET)]. Like Asn-containing proteins and polypeptides, polyaspartamides undergo uncatalyzed degradation under model physiological conditions (10 mM phosphate buffer, pH 7.4, 150 mM NaCl). We studied the uncatalyzed degradation of the polyaspartamide polymers by size exclusion chromatography and found that the degradation rate was sensitive to the nature of the pendant groups. The metal-free polymer underwent somewhat slower degradation than the corresponding polymers in which the DTPA groups were saturated with Eu(3+) or In(3+), but even after 14 days, substantial fractions of the polymers survived. We conclude that these polymers undergo negligible degradation on the time scale (24-48 h) of radioimmunotherapy treatment of tumors with (111)In. From a mechanistic perspective, we note that these degradation rates are on the order of the deamidation rates reported [J. Peptide Res. 2004, 63, 426] for Asn-containing pentapeptides, with half-times on the order of 10 days, but much slower than the rapid decay (hours) reported recently [Biomaterials 2010, 31, 3707] for poly{N'-[N-(2-aminoethyl)-2-aminoethyl]aspartamide} itself. This variation in degradation rate can be explained in terms of the influence of positive charges on the pendant group enhancing the acidity of the side-chain amide nitrogen of the aspartamide repeat unit. The DET pendant group is positively charged at pH 7, but in indium-loaded PAsp(DTPA) this charge is offset by the net negative charge of the DTPA-In complex.     

Remarkable stability of the proton translocating F1FO-ATP synthase from the thermophilic cyanobacterium Thermosynechococcus elongatus BP-1

For functional characterization, we isolated the F1FO-ATP synthase of the thermophilic cyanobacterium Thermosynechococcus elongatus. Because of the high content of phycobilisomes, a combination of dye-ligand chromatography and anion exchange chromatography was necessary to yield highly pure ATP synthase. All nine single F1FO subunits were identified by mass spectrometry. Western blotting revealed the SDS stable oligomer of subunits c in T. elongatus. In contrast to the mass archived in the database (10,141 Da), MALDI-TOF-MS revealed a mass of the subunit c monomer of only 8238 Da. A notable feature of the ATP synthase was its ability to synthesize ATP in a wide temperature range and its stability against chaotropic reagents. After reconstitution of F1FO into liposomes, ATP synthesis energized by an applied electrochemical proton gradient demonstrated functional integrity. The highest ATP synthesis rate was determined at the natural growth temperature of 55 degrees C, but even at 95 degrees C ATP production occurred. In contrast to other prokaryotic and eukaryotic ATP synthases which can be disassembled with Coomassie dye into the membrane integral and the hydrophilic part, the F1FO-ATP synthase possessed a particular stability. Also with the chaotropic reagents sodium bromide and guanidine thiocyanate, significantly harsher conditions were required for disassembly of the thermophilic ATP synthase.     

Activity of neuromodulatory neurones during stepping of a single insect leg

Octopamine plays a major role in insect motor control and is released from dorsal unpaired median (DUM) neurones, a group of cells located on the dorsal midline of each ganglion. We were interested whether and how these neurones are activated during walking and chose the semi-intact walking preparation of stick insects that offers to investigate single leg-stepping movements. DUM neurones were characterized in the thoracic nerve cord by backfilling lateral nerves. These backfills revealed a population of 6-8 efferent DUM cells per thoracic segment. Mesothoracic DUM cells were subsequently recorded during middle leg stepping and characterized by intracellular staining. Seven out of eight identified individual different types of DUM neurones were efferent. Seven types except the DUMna nl2 were tonically depolarized during middle leg stepping and additional phasic depolarizations in membrane potential linked to the stance phase of the middle leg were observed. These DUM neurones were all multimodal and received depolarizing synaptic drive when the abdomen, antennae or different parts of the leg were mechanically stimulated. We never observed hyperpolarising synaptic inputs to DUM neurones. Only one type of DUM neurone, DUMna, exhibited spontaneous rhythmic activity and was unaffected by different stimuli or walking movements.     

Dandelion Seed Dispersal: The Horizontal Wind Speed Does Not Matter for Long-Distance Dispersal - it is Updraft!

Abstract: Long-distance dispersal of seeds (LDD) surely affects most ecological and evolutionary processes related to plant species. Hence, numerous attempts to quantify LDD have been made and, especially for wind dispersal, several simulation models have been developed. However, the mechanisms promoting LDD by wind still remain ambiguous and the effects of different weather conditions on LDD, although recognized as important, have only rarely been investigated. Here we examine the influence of wind speed and updrafts on dispersal of dandelion ( Taraxacum officinale agg.), a typical wind-dispersed herb of open habitats. We used PAPPUS, a weather-sensitive mechanistic simulation model of wind dispersal, which considers frequency distribution of weather conditions during the period the simulation refers to. A simulation for the 4-month shedding period of dandelion shows that high wind speed does not promote LDD. In contrast, vertical turbulence, especially convective updrafts, are of overwhelming importance. Mainly caused by updrafts, in the simulations more than 0.05 % of dandelion seeds were dispersed beyond 100 m, a distance commonly used to define LDD. We conclude that long-distance dispersal of seeds of herbaceous species with falling velocities < 0.5 - 1.0 ms^-1 is mainly caused by convective updrafts.     

Oxygen isotope fractionations across individual leaf carbohydrates in grass and tree species

Almost no δ¹⁸O data are available for leaf carbohydrates, leaving a gap in the understanding of the δ¹⁸O relationship between leaf water and cellulose. We measured δ¹⁸O values of bulk leaf water (δ¹⁸O_LW) and individual leaf carbohydrates (e.g. fructose, glucose and sucrose) in grass and tree species and δ¹⁸O of leaf cellulose in grasses. The grasses were grown under two relative humidity (rH) conditions. Sucrose was generally ¹⁸O‐enriched compared with hexoses across all species with an apparent biosynthetic fractionation factor (ε_bio) of more than 27‰ relative to δ¹⁸O_LW, which might be explained by isotopic leaf water and sucrose synthesis gradients. δ¹⁸O_LW and δ¹⁸O values of carbohydrates and cellulose in grasses were strongly related, indicating that the leaf water signal in carbohydrates was transferred to cellulose (ε_bio = 25.1‰). Interestingly, damping factor p_exp_x, which reflects oxygen isotope exchange with less enriched water during cellulose synthesis, responded to rH conditions if modelled from δ¹⁸O_LW but not if modelled directly from δ¹⁸O of individual carbohydrates. We conclude that δ¹⁸O_LW is not always a good substitute for δ¹⁸O of synthesis water due to isotopic leaf water gradients. Thus, compound‐specific δ¹⁸O analyses of individual carbohydrates are helpful to better constrain (post‐)photosynthetic isotope fractionation processes in plants.     

Bacterial membrane proteomics

About one quarter to one third of all bacterial genes encode proteins of the inner or outer bacterial membrane. These proteins perform essential physiological functions, such as the import or export of metabolites, the homeostasis of metal ions, the extrusion of toxic substances or antibiotics, and the generation or conversion of energy. The last years have witnessed completion of a plethora of whole-genome sequences of bacteria important for biotechnology or medicine, which is the foundation for proteome and other functional genome analyses. In this review, we discuss the challenges in membrane proteome analysis, starting from sample preparation and leading to MS-data analysis and quantification. The current state of available proteomics technologies as well as their advantages and disadvantages will be described with a focus on shotgun proteomics. Then, we will briefly introduce the most abundant proteins and protein families present in bacterial membranes before bacterial membrane proteomics studies of the last years will be presented. It will be shown how these works enlarged our knowledge about the physiological adaptations that take place in bacteria during fine chemical production, bioremediation, protein overexpression, and during infections. Furthermore, several examples from literature demonstrate the suitability of membrane proteomics for the identification of antigens and different pathogenic strains, as well as the elucidation of membrane protein structure and function.     

Improved Identification of Membrane Proteins by MALDI-TOF MS/MS Using Vacuum Sublimated Matrix Spots on an Ultraphobic Chip Surface

Integral membrane proteins are notoriously difficult to identify and analyze by mass spectrometry because of their low abundance and limited number of trypsin cleavage sites. Our strategy to address this problem is based on a novel technology for MALDI-MS peptide sample preparation that increases the success rate of membrane protein identification by increasing the sensitivity of the MALDI-TOF system. For this, we used sample plates with predeposited matrix spots of CHCA crystals prepared by vacuum sublimation onto an extremely low wettable (ultraphobic) surface. In experiments using standard peptides, an up to 10-fold gain of sensitivity was found for on-chip preparations compared with classical dried-droplet preparations on a steel target. In order to assess the performance of the chips with membrane proteins, three model proteins (bacteriorhodopsin, subunit IV(a) of ATP synthase, and the cp47 subunit from photosystem II) were analyzed. To mimic realistic analysis conditions, purified proteins were separated by SDS-PAGE and digested with trypsin. The digest MALDI samples were prepared either by dried-droplet technique on steel plates using CHCA as matrix, or applied directly onto the matrix spots of the chip surface. Significantly higher signal-to-noise ratios were observed for all of the spectra resulting from on-chip preparations of different peptides.In a second series of experiments, the membrane proteome of Rhodococcus jostii RHA1 was investigated by AIEC/SDS-PAGE in combination with MALDI-TOF MS/MS. As in the first experiments, Coomassie-stained SDS-PAGE bands were digested and the two different preparation methods were compared. For preparations on the Mass·Spec·Turbo Chip, 43 of 60 proteins were identified, whereas only 30 proteins were reliably identified after classical sample preparation. Comparison of the obtained Mascot scores, which reflect the confidence level of the protein identifications, revealed that for 70% of the identified proteins, higher scores were obtained by on-chip sample preparation. Typically, this gain was a consequence of higher sequence coverage due to increased sensitivity.     

Effects of environmental parameters, leaf physiological properties and leaf water relations on leaf water delta18O enrichment in different Eucalyptus species

Stable oxygen isotope ratios (delta18O) have become a valuable tool in the plant and ecosystem sciences. The interpretation of delta18O values in plant material is, however, still complicated owing to the complex interactions among factors that influence leaf water enrichment. This study investigated the interplay among environmental parameters, leaf physiological properties and leaf water relations as drivers of the isotopic enrichment of leaf water across 17 Eucalyptus species growing in a common garden. We observed large differences in maximum daily leaf water delta18O across the 17 species. By fitting different leaf water models to these empirical data, we determined that differences in leaf water delta18O across species are largely explained by variation in the Péclet effect across species. Our analyses also revealed that species-specific differences in transpiration do not explain the observed differences in delta18O while the unconstrained fitting parameter 'effective path length' (L) was highly correlated with delta18O. None of the leaf morphological or leaf water related parameters we quantified in this study correlated with the L values we determined even though L was typically interpreted as a leaf morphological/anatomical property. A sensitivity analysis supported the importance of L for explaining the variability in leaf water delta18O across different species. Our investigation highlighted the importance of future studies to quantify the leaf properties that influence L. Obtaining such information will significantly improve our understanding of what ultimately determines the delta18O values of leaf water across different plant species.     

Occurrence and prevalence of Clostridium perfringens in polar bears from Svalbard, Norway

To obtain insight into the occurrence and prevalence of Clostridium perfringens and its major toxins in polar bears (Ursus maritimus), we took fecal samples for bacteriologic analysis from live-captured bears in the Svalbard Archipelago, Norway, in 2001. Clostridium perfringens was isolated from 40 of 92 samples (44%). Thirty strains were further characterized by determining toxin type and were classified to be type A, while one was also positive for the gene encoding beta2-toxin. Despite the fact that C. perfringens type A has been associated with fatal diseases in several animal species as well as in humans, our data indicate that C. perfringens type A is an normal inhabitant of the gastrointestinal tract of polar bears.     

Nitration of the Striatal Na,K-ATPase α3 Isoform Occurs in Normal Brain Development but Is Not Increased During Hypoxia-Ischemia in Newborn Piglets

Neonatal hypoxia-ischemia (HI) can result in significant sensorimotor abnormalities, including movement and posture disorders. These neurological impairments are believed to result from basal ganglia (striatum) damage, but the exact cause of this injury is not known. One mechanism involved in brain injury after HI is the generation of reactive oxygen species, which damage cellular macromolecules. We tested the hypothesis that inactivation of plasma membrane enzyme Na,K-ATPase during striatal neurodegeneration after HI emerges with peroxynitrite attack on the enzyme. In vitro, reaction of peroxynitrite (100–500 M) with purified Na,K-ATPase produced nitration of the (catalytic) and (transport) subunits, as quantified by immunoblots of the reaction products for nitrotyrosine. To evaluate for peroxynitrite damage to Na,K-ATPase in vivo, striatal plasma membrane fractions from 1-week-old piglets subjected to asphyxic cardiac arrest and recovery were also studied by immunoprecipitation. During the progression of striatal neurodegeneration and loss of enzyme function 3–24 h after arrest, nitration of the ₃ (neuronal) isoform of Na,K-ATPase was not increased relative to sham control. Suprisingly, however, nitration of this isoform occurs during normal brain development and peaks at 2 weeks of age. We conclude that Na,K-ATPase is a target of peroxynitrite, but that this mechanism is not responsible for enzyme inactivation after HI. Protein nitration may serve as marker of other normal, noninjurious cell processes in the developing brain.