Ice-free cryopreservation of heart valve allografts: better extracellular matrix preservation in vivo and preclinical results

The purpose of this study was evaluation of an ice-free cryopreservation method for heart valves in an allogeneic juvenile pulmonary sheep implant model and comparison with traditionally frozen cryopreserved valves. Hearts of 15 crossbred Whiteface sheep were procured in Minnesota. The valves were processed in South Carolina and the pulmonary valves implanted orthotopically in 12 black faced Heidschnucke sheep in Germany. The ice-free cryopreserved valves were cryopreserved in 12.6?mol/l cryoprotectant (4.65, 4.65, and 3.31?mol/l of dimethylsulfoxide, formamide and 1,2-propanediol) and stored at ?80°C. Frozen valves were cryopreserved by controlled slow rate freezing in 1.4?mol/l dimethylsulfoxide and stored in vapor-phase nitrogen. Aortic valve tissues were used to evaluate the impact of preservation without implantation. Multiphoton microscopy revealed reduced but not significantly damaged extracellular matrix before implantation in frozen valves compared with ice-free tissues. Viability assessment revealed significantly less metabolic activity in the ice-free valve leaflets and artery samples compared with frozen tissues (P?<?0.05). After 3 and 6?months in vivo valve function was determined by two-dimensional echo-Doppler and at 7?months the valves were explanted. Severe valvular stenosis with right heart failure was observed in recipients of frozen valves, the echo data revealed increased velocity and pressure gradients compared to ice-free valve recipients (P?=?0.0403, P?=?0.0591). Histo-pathology showed significantly thickened leaflets in the frozen valves (P?<?0.05) and infiltrating CD3+ T-cells (P?<?0.05) compared with ice-free valve leaflets. Multiphoton microscopy at explant revealed reduced inducible autofluorescence and extracellular matrix damage in the frozen explants and well preserved structures in the ice-free explant leaflets. In conclusion, ice-free cryopreservation of heart valve transplants at ?80°C avoids ice formation, tissue-glass cracking and preserves extracellular matrix integrity resulting in minimal inflammation and improved hemodynamics in allogeneic juvenile sheep.     

Torque generation of kinesin motors is governed by the stability of the neck domain

In long-range transport of cargo, prototypical kinesin-1 steps along a single protofilament on the microtubule, an astonishing behavior given the number of theoretically available binding sites on adjacent protofilaments. Using a laser trap assay, we analyzed the trajectories of several representatives from the kinesin-2 class on freely suspended microtubules. In stark contrast to kinesin-1, these motors display a wide range of left-handed spiraling around microtubules and thus generate torque during cargo transport. We provide direct evidence that kinesin's neck region determines the torque-generating properties. A model system based on kinesin-1 corroborates this result: disrupting the stability of the neck by inserting flexible peptide stretches resulted in pronounced left-handed spiraling. Mimicking neck stability by crosslinking significantly reduced the spiraling of the motor up to the point of protofilament tracking. Finally, we present a model that explains the physical basis of kinesin's spiraling around the microtubule.     

Ectopic expression of Arabidopsis thaliana plasma membrane intrinsic protein 2 aquaporins in lily pollen increases the plasma membrane water permeability of grain but not of tube protoplasts

To investigate the role of aquaporin-mediated water transport during pollen grain germination and tube growth, Arabidopsis thaliana plasma membrane intrinsic proteins (PIPs) were expressed in pollen of Lilium longiflorum (lily). Successful expression of AtPIPs in particle-bombarded lily pollen grains was monitored by co-expression with fluorescent proteins and single-cell RT-PCR, and by measuring the water permeability coefficient (P(os)) in swelling assays using protoplasts prepared from transformed pollen grains and tubes. Expression of AtPIP1;1 and AtPIP1;2 in pollen grains resulted in P(os) values similar to those measured in nontransformed pollen grain protoplasts (6.65 +/- 2.41 microm s(-1)), whereas expression of AtPIP2 significantly increased P(os) (AtPIP2;1, 13.79 +/- 6.38; AtPIP2;2, 10.16 +/- 3.30 microm s(-1)). Transformation with combinations of AtPIP1 and AtPIP2 did not further enhance P(os). Native pollen tube protoplasts showed higher P(os) values (13.23 +/- 4.14 microm s(-1)) than pollen grain protoplasts but expression of AtPIP2;1 (18.85 +/- 7.60 microm s(-1)) did not significantly increase their P(os) values. Expression of none of the tested PIPs had any effect on pollen tube growth rates. The ectopic expression of AtPIP2s in lily pollen increased the water permeability of the plasma membrane in pollen grains, but not in pollen tubes. The measured endogenous water permeability does not limit water uptake during tube growth, but has to be regulated to prevent tube bursting.     

Analysis of immunoglobulin glycosylation by LC-ESI-MS of glycopeptides and oligosaccharides

Two LC-ESI-MS methods for the analysis of antibody glycosylation are presented. In the first approach, tryptic glycopeptides are separated by RP chromatography and analyzed by ESI-MS. This "glycopeptide strategy" allows a protein- and subclass-specific quantitation of both neutral and sialylated glycan structures. Additional information about under- or deglycosylation and the protein backbone, e.g., termini, can be extracted from the same data. In the second LC-ESI-MS method, released oligosaccharides are separated on porous graphitic carbon (PGC). A complete structural assignment of neutral and sialylated oligosaccharides occurring on antibodies is thereby achieved in one chromatographic run. The two methods were applied to polyclonal human IgG, to commercial mAb expressed in CHO cells (Rituximab, Xolair, and Herceptin), in SP2/0 (Erbitux and Remicade) or NS0 cells (Zenapax) and the anti-HIV antibody 4E10 produced either in CHO cells or in a human cell line. Both methods require comparably little sample preparation and can be applied to SDS-PAGE bands. They both outperform non-MS methods in terms of reliability of peak assignment and MALDI-MS of underivatized glycans with regard to the recording of sialylated structures. Regarding fast and yet detailed structural assignment, LC-MS on graphitic carbon supersedes all other current methods.     

Direct methods and residue type specific isotope labeling in NMR structure determination and model-driven sequential assignment

Direct methods in NMR based structure determination start from an unassigned ensemble of unconnected gaseous hydrogen atoms. Under favorable conditions they can produce low resolution structures of proteins. Usually a prohibitively large number of NOEs is required, to solve a protein structure ab-initio, but even with a much smaller set of distance restraints low resolution models can be obtained which resemble a protein fold. One problem is that at such low resolution and in the absence of a force field it is impossible to distinguish the correct protein fold from its mirror image. In a hybrid approach these ambiguous models have the potential to aid in the process of sequential backbone chemical shift assignment when ¹³C^β and ¹³C′ shifts are not available for sensitivity reasons. Regardless of the overall fold they enhance the information content of the NOE spectra. These, combined with residue specific labeling and minimal triple-resonance data using ¹³C^α connectivity can provide almost complete sequential assignment. Strategies for residue type specific labeling with customized isotope labeling patterns are of great advantage in this context. Furthermore, this approach is to some extent error-tolerant with respect to data incompleteness, limited precision of the peak picking, and structural errors caused by misassignment of NOEs. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Electronic wiring of a multi-redox site membrane protein in a biomimetic surface architecture

Bioelectronic coupling of multi-redox-site membrane proteins was accomplished with cytochrome c oxidase (CcO) as an example. A biomimetic membrane system was used for the oriented immobilization of the CcO oxidase on a metal electrode. When the protein is immobilized with the CcO binding side directed toward the electrode and reconstituted in situ into a lipid bilayer, it is addressable by direct electron transfer to the redox centers. Electron transfer to the enzyme via the spacer, referred to as electronic wiring, shows an exceptionally high rate constant. This allows a kinetic analysis of all four consecutive electron transfer steps within the enzyme to be carried out. Electron transfer followed by rapid scan cyclic voltametry in combination with surface-enhanced resonance Raman spectroscopy provides mechanistic and structural information about the heme centers. Probing the enzyme under turnover conditions showed mechanistic insights into proton translocation coupled to electron transfer. This bioelectronic approach opens a new field of activity to investigate complex processes in a wide variety of membrane proteins.     

The biology of non-weedy hemiparasiticOrobanchaceae: Synthesis and perspectives

This paper gives an overview of current research into the biology of hemiparasiticOrobanchaceae, formerly part of theScrophulariaceae. It is based on presentations and discussions that took place during the First International Symposium on non-weedy hemiparasiticOrobanchaceae in April 2004 in Wageningen. Aspects such as taxonomy and evolution, ecophysiology, population and restoration ecology are discussed, thus identifying challenges for future research. Hemiparasites have very different life histories, and the robust molecular phylogeny will now permit testing hypotheses regarding the evolution of these life histories, degree of parasitism and host specialization. In a number of genera, evolution is in full swing, leading to taxonomical complications, but at the same time offering opportunities for phylogeographical research. In ecophysiology, the challenge is to better understand what makes a good host and to investigate further the chemical signals emitted by the host and their use in regulating parasite development. Finally, the results of sowing hemiparasites to speed up the restoration of nutrient-poor grasslands are still very variable, and we need a more thorough understanding of the factors influencing population dynamics, which should also enable us to devise better management plans for threatened hemiparasitic species.     

Ex vivo gene transfer in the years to come

Synovial fibroblasts (SFs) have become a major target for ex vivo gene transfer in rheumatoid arthritis (RA), but efficient transduction of RA-SFs still is a major problem. The low proliferation rate and heterogeneity of RA-SFs, together with their lack of highly specific surface receptors, have hampered a more extensive application of this technique. Improving transduction protocols with conventional viral vectors, therefore, as well as developing novel strategies, such as alternative target cells, and novel delivery systems constitute a major challenge. Recent progress in this field will lead to the achievement of high transgene expression, and will facilitate the use of gene transfer in human trials.     

Fe homeostasis in plant cells: Does nicotianamine play multiple roles in the regulation of cytoplasmic Fe concentration?

The cellular and intracellular localization of the non-proteogenic amino acid nicotianamine (NA) in leaves and root elongation zones was immunochemically investigated in pea (Pisum sativum L.) and tomato (Lycopersicon esculentum Mill.) plants grown under various iron regimes and in three mutants defective in the regulation of iron uptake. Strongest immunostaining was observed in the over-accumulating pea mutants brz and dgl, and in iron-loaded wild-type plants. Fe concentration and NA level paralleled staining intensity, indicating that NA synthesis is induced by high iron availability. While label was mainly present in the cytoplasm under normal (10 microM) Fe supply and under Fe deprivation, most of the labeling was present in the vacuole in iron-loaded plants. This pattern resembled the distribution of NA in Fe over-accumulating mutants, indicating the possible importance of vacuolar sequestration in the detoxification of excess Fe. Based on the dependence of the cellular distribution of NA on the iron nutritional status of the plant, a possible role of NA in buffering free Fe in root and leaf cells was inferred. We show here for the first time that the NA concentration is increased in response to iron overload, indicating that, besides other classes of intracellular metal-binding ligands, NA may play an essential role in iron tolerance.     

Seasonal Variation of Diurnal and Ultradian Rhythms in Red Deer

To describe the normal rhythmic behavioural patterns and to test procedures for the detection of short-time disturbances, 4 red deer were studied in 2 quasi-natural enclosures. Activity and feeding were recorded by means of the storage-telemetrysystem ETHOSYS®. Daily and monthly mean values, power spectra and 'Degrees of Functional Coupling' (DFC) were calculated. DFC were applied to measure stability and harmonic synchronisation between ultradian rhythms and the 24-hours period. The general patterns of activity and feeding were nearly identical in all animals and closely related to photoperiod change. Levels of total activity and feeding were lowest in winter and highest in summer. In winter highest activity was generally observed in daylight hours and shifted gradually to dark hours in summer. Spectral analysis of activity and feeding revealed a time pattern in which ultradian components, between 4.8 and 12 hrs in period length, mostly exceeded the diurnal rhythm power. Compared to winter higher frequencies were found in activity and feeding in spring, summer and autumn. During such periods up to 8 strong bouts of activity per day (3-hrs rhythm) were observed. These rhythmic patterns are discussed in the context of red deer feeding strategy. Short-term disturbances by humans and changing feeding conditions resulted in lowered DFCs. Time pattern analysis of long-term and continuously measured behavioural parameters proved to be an appropriate approach for observing general living conditions and for detecting disturbances.