The evolutionary history of the white-rayed species of Melampodium (Asteraceae) involved multiple cycles of hybridization and polyploidization

? Premise of the study: Polyploidy plays an important role in race differentiation and eventually speciation. Underlying mechanisms include chromosomal and genomic changes facilitating reproductive isolation and/or stabilization of hybrids. A prerequisite for studying these processes is a sound knowledge on the origin of polyploids. A well-suited group for studying polyploid evolution consists of the three species of Melampodium ser. Leucantha (Asteraceae): M. argophyllum, M. cinereum, and M. leucanthum. ? Methods: The origin of polyploids was inferred using network and tree-based phylogenetic analyses of several plastid and nuclear DNA sequences and of fingerprint data (AFLP). Genome evolution was assessed via genome size measurements, karyotype analysis, and in situ hybridization of ribosomal DNA. ? Key results: Tetraploid cytotypes of the phylogenetically distinct M. cinereum and M. leucanthum had, compared to the diploid cytotypes, doubled genome sizes and no evidence of gross chromosomal rearrangements. Hexaploid M. argophyllum constituted a separate lineage with limited intermixing with the other species, except in analyses from nuclear ITS. Its genome size was lower than expected if M. cinereum and/or M. leucanthum were involved in its origin, and no chromosomal rearrangements were evident. ? Conclusions: Polyploids in M. cinereum and M. leucanthum are of recent autopolyploid origin in line with the lack of significant genomic changes. Hexaploid M. argophyllum also appears to be of autopolyploid origin against the previous hypothesis of an allopolyploid origin involving the other two species, but some gene flow with the other species in early phases of differentiation cannot be excluded.     

Characterization and in vitro immunomodulatory screening of fructo-oligosaccharides of Asparagus racemosus Willd

Asparagus racemosus Linn. (Fam. Liliaceae) is an ethno-pharmacologically acclaimed Ayurvedic medicinal plant. In the present study, aqueous extract of A. racemosus (ARC) was fractionated and screened for the polysaccharide fraction (ARP). The characterization was done by enzymatic, Size Exclusion, gas chromatography with flame ionization detector (GC-FID), high pressure anion exchange chromatography (HPAEC) and thin layer chromatographic analyses. Phyto-chemical evaluation confirmed the presence of 26.7% of 2 → 1 linked fructo-oligosaccharides (FOS). They have a degree of polymerization (DP) of nearly 7-8. Cytotoxicity evaluation on P388 cell lines was consistent with low cytotoxicity of the extracts. In vitro Natural Killer (NK) cell activity was evaluated using human peripheral blood mononuclear cells (PBMC) isolated from whole blood on a ficoll-hypaque density gradient. K562 a myeloid leukemia cell line, were used as target cells. ARC, tested over the range 0.2-50 μg/ml, showed a dose-related stimulation of NK cell activity with a peak increase of 16.9 ± 4.4% at 5.6 μg/ml. However, ARP demonstrated a higher stimulatory activity of 51.8 ± 1.2% at 25 μg/ml. The results indicate that the FOS from A. racemosus potentiates the NK cell activity and this could be an important mechanism underpinning the ‘Rasayana’ properties of this plant.     

Flavodoxin displays dose-dependent effects on photosynthesis and stress tolerance when expressed in transgenic tobacco plants

Ferredoxins are iron–sulfur proteins involved in various one-electron transfer pathways. Ferredoxin levels decrease under adverse environmental conditions in photosynthetic organisms. In cyanobacteria, this decline is compensated by induction of flavodoxin, an isofunctional flavoprotein. Flavodoxin is not present in higher plants, but transgenic Nicotiana tabacum lines accumulating Anabaena flavodoxin in plastids display increased tolerance to different sources of environmental stress. As the degree of tolerance correlated with flavodoxin dosage in plastids of nuclear-transformed transgenic tobacco, we prepared plants expressing even higher levels of flavodoxin by direct plastid transformation. A suite of nuclear- and chloroplast-transformed lines expressing a wide range of flavodoxin levels, from 0.3 to 10.8?μmol?m^?2, did not exhibit any detectable growth phenotype relative to the wild type. In the absence of stress, the contents of both chlorophyll a and carotenoids, as well as the photosynthetic performance (photosystem II maximum efficiency, photosystem II operating efficiency, electron transport rates and carbon assimilation rates), displayed a moderate increase with flavodoxin concentrations up to 1.3–2.6?μmol flavodoxin m^?2, and then declined to wild-type levels. Stress tolerance, as estimated by the damage inflicted on exposure to the pro-oxidant methyl viologen, also exhibited a bell-shaped response, with a significant, dose-dependent increase in tolerance followed by a drop in the high-expressing lines. The results indicate that optimal photosynthetic performance and stress tolerance were observed at flavodoxin levels comparable to those of endogenous ferredoxin. Further increases in flavodoxin content become detrimental to plant fitness.     

A European phylogeography of Rhinanthus minor compared to Rhinanthus angustifolius: unexpected splits and signs of hybridization

Rhinanthus minor and Rhinanthus angustifolius (Orobanchaceae) are annual hemiparasites, which occur sympatrically in Europe and are known to hybridize. We studied chloroplast and nuclear (amplified fragment length polymorphism [AFLP]) diversity in R. minor and compared genetic structuring in this species with R. angustifolius by analyzing the AFLP data for both species simultaneously. The AFLP data revealed that populations in Italy, Greece, and southeast Russia initially identified as R. minor were so distant from the other R. minor populations that they probably belong to another, yet unidentified taxon, and we refer to them as Rhinanthus sp. R. minor s.s. showed a clear geographic genetic structure in both the chloroplast DNA (cpDNA) and nuclear genome. The simultaneous analysis of both species shed new light on the previously published findings for R. angustifolius, because some populations now turned out to belong to R. minor. The admixture analysis revealed very few individuals of mixed R. minor-R.angustifolius ancestry in the natural populations in the west of Europe, while admixture levels were higher in the east. The combined haplotype network showed that haplotype H1 was shared among all species and is likely to be ancestral. H2 was more abundant in R. angustifolius and H3 in R. minor, and the latter probably arose from H1 in this species in the east of Europe. The occurrence of H3 in R. angustifolius may be explained by introgression from R. minor, but without interspecific admixture, these are likely to have been old hybridization events. Our study underlines the importance of including related species in phylogeographic studies.     

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.     

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.     

Lipidosislike renal changes in rats treated with chlorphentermine or with tricyclic antidepressants

Histological, histochemical and ultrastructural examinations were performed on renal tissues of rats after prolonged oral administration of the anorectic drug chlorphentermine or of the tricyclic antidepressants iprindole, imipramine and clomipramine. All drugs caused the formation of multilamellated cytoplasmic inclusions throughout the nephron and the collecting duct system, and in interstitial cells. The cytological alterations were most prominent in the glomerular podocytes, in the proximal convoluted tubules, and in the collecting duct system. In view of the histochemical properties (staining with Baker's acid hematein) and the ultrastructural appearance of the cytoplasmic inclusions the cellular alterations are interpreted as a renal manifestation of a generalized lipidosis induced by drugs of amphiphilic character.