Isolation of Osteoprogenitors from Human Jaw Periosteal Cells: A Comparison of Two Magnetic Separation Methods

Human jaw periosteum tissue contains osteoprogenitors that have potential for tissue engineering applications in oral and maxillofacial surgeries. To isolate osteoprogenitor cells from heterogeneous cell populations, we used the specific mesenchymal stem cell antigen-1 (MSCA-1) antibody and compared two magnetic separation methods. We analyzed the obtained MSCA-1⁺ and MSCA-1⁻ fractions in terms of purity, yield of positive/negative cells and proliferative and mineralization potentials. The analysis of cell viability after separation revealed that the EasySep method yielded higher viability rates, whereas the flow cytometry results showed a higher purity for the MACS-separated cell fractions. The mineralization capacity of the osteogenic induced MSCA-1⁺ cells compared with the MSCA-1⁻ controls using MACS was 5-fold higher, whereas the same comparison after EasySep showed no significant differences between both fractions. By analyzing cell proliferation, we detected a significant difference between the proliferative potential of the osteogenic cells versus untreated cells after the MACS and EasySep separations. The differentiated cells after MACS separation adjusted their proliferative capacity, whereas the EasySep-separated cells failed to do so. The protein expression analysis showed small differences between the two separation methods. Our findings suggest that MACS is a more suitable separation method to isolate osteoprogenitors from the entire jaw periosteal cell population.     

Serum- and albumin-free cryopreservation of endothelial monolayers with a new solution

Cryopreservation is the only long-term storage option for the storage of vessels and vascular constructs. However, endothelial barrier function is almost completely lost after cryopreservation in most established cryopreservation solutions. We here aimed to improve endothelial function after cryopreservation using the 2D-model of porcine aortic endothelial cell monolayers.?The monolayers were cryopreserved in cell culture medium or cold storage solutions based on the 4°C vascular preservation solution TiProtec^®, all supplemented with 10% DMSO, using different temperature gradients. After short-term storage at ?80°C, monolayers were rapidly thawed and re-cultured in cell culture medium.?Thawing after cryopreservation in cell culture medium caused both immediate and delayed cell death, resulting in 11 ± 5% living cells after 24 h of re-culture. After cryopreservation in TiProtec and chloride-poor modifications thereof, the proportion of adherent viable cells was markedly increased compared to cryopreservation in cell culture medium (TiProtec: 38 ± 11%, modified TiProtec solutions ≥ 50%). Using these solutions, cells cryopreserved in a sub-confluent state were able to proliferate during re-culture. Mitochondrial fragmentation was observed in all solutions, but was partially reversible after cryopreservation in TiProtec and almost completely reversible in modified solutions within 3 h of re-culture. The superior protection of TiProtec and its modifications was apparent at all temperature gradients; however, best results were achieved with a cooling rate of ?1°C/min.?In conclusion, the use of TiProtec or modifications thereof as base solution for cryopreservation greatly improved cryopreservation results for endothelial monolayers in terms of survival and of monolayer and mitochondrial integrity.     

Gradation in Nutrient Composition and Photosynthetic Pathways Across the Restinga Vegetation of Brazil

The restinga comprises coastal vegetation formations which dominate the Atlantic seaboard of Brazil. Exposed sand ridges and associated lagoon systems have poorly developed soils subject to pronounced water deficits. Distinct vegetation zones support a high diversity of life forms, and a comparative study has been undertaken to investigate interactions between degree of exposure, nutrient supply and photosynthetic pathway (C₃, or CAM) in selected species across the restinga. A number of species occurring throughout the restinga were chosen as representative species of different life forms, comprising C₃ pioneer shrubs (Eugenia rotundifolia and Erythroxylum ovalifolium), impounding (tank) terrestrial bromeliad (Neoregelia cruenta: CAM) and the atmospheric epiphyte (Tillandsia stricta: CAM). Comparisons of plant and soil nutrient composition, and airborne deposition were conducted for each zone. Soil nutrient content and organic matter were closely related, reaching a maximum in zone 4, the seaward face of the inner dune. Salt concentration in leaves was independent of atmospheric deposition for the terrestrial species, in contrast to the atmospheric epiphyte T. stricta. In the slack area, vegetation formed characteristic “islands” with the soil beneath enriched in nutrients, suggesting a complex interplay between plants and soil during the development of vegetation succession. Here, two additional trees were investigated, C₃ and CAM members of the Clusiaceae, respectively Clusia lanceolata and C. fluminensis. Stable isotope composition of nitrogen (δ¹⁵N) was generally more negative (depleted in ¹⁵N) in plants with low total nitrogen content. This was exemplified by the atmospheric bromeliad, T. stricta, with an N content of 2.91 g/kg and δ¹⁵N of ?12.3 per mil. Stable isotopes of carbon (δ¹³C) were used to identify the distribution of photosynthetic pathways, and while the majority of bromeliads and orchids were CAM, analysis of the soil organic matter suggested that C₃ plants made the major contribution in each zone of the restinga. Since δ¹³C of plant material also suggested that water supply was optimal in zone 4, we conclude that succession and high diversity in the restinga is dependent on exposure, edaphic factors, and perhaps a critical mass of vegetation required to stabilize nutrient relations of the system.     

Induktion der Phenylalanin-Ammonium-Lyase in Gewebekulturen vonHaplopappus gracilis

Zusammenfassung In Zellkulturen vonHaplopappus gracilis, die nur im Licht Anthocyan synthetisierten, wurde die PAL-Aktivität untersucht. Diese Kulturen wiesen auch im Dunkeln, abhängig vom Zeitpunkt der letzten Übertragung auf frisches Nährmedium, deutliche Unterschiede in der Höhe der PAL-Aktivität auf. Blaulicht führte in jedem Falle zu einer Erhöhung der PAL-Aktivität und zu anschließender Anthocyansynthese. Den größten Einfluß auf die Aktivität des Enzyms hatte Blaulicht auf 3 Wochen alten Kulturen. Nach 48stündiger Bestrahlung betrug der Anstieg der Enzymaktivität 400%, dann folgte ein allmählicher Rückgang. Der Proteingehalt und die MDH-Aktivität der Kulturen blieben während dieser Zeit konstant. Ein PAL-inaktivierendes System konnte in denHaplopappuskulturen nicht nachgewiesen werden. Aber Actinomycin D und Puromycin hemmten die Blaulicht-induzierte PAL-Synthese. Rotlicht hatte in allen Versuchen keinen Effekt auf die Höhe der Enzymaktivität.

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Induction of phenylalanine ammonia-lyase in tissue cultures ofHaplopappus gracilis

Summary The PAL activity of cell cultures ofHaplopappus gracilis was investigated. These showed anthocyanin synthesis only after exposure to light. Blue light always led to an increase of PAL activity and subsequent anthocyanin production. But also in the dark, only depending on the time after the passage to fresh medium significant changes in PAL activity have been detected. The highest effect of blue light on the activity of the enzyme was observed in the cultures three weeks after passage to fresh medium. Irradiation for 48 hours with blue light then resulted in a 400% rise of PAL activity followed by a gradual decline. In these experiments no changes of total protein content or MDH activity were found. There also was no evidence for a PAL-inactivating system. Actinomycin D and Puromycin inhibited the blue light induced increase of PAL activity. Irradiation with red resp. farred light alone or given after induction with blue light had no effect on the enzyme synthesis.

     

Chloroplastenvermehrung in kernlosen Teilstücken vonAcetabularia mediterranea undAcetabularia cliftonii und ihre Abhängigkeit von inneren Faktoren

Zusammenfassung In kernlosen, wachsenden Zellfragmenten (Vorderstücke) vonAcetabularia mediterranea undAcetabularia (Polyphysa) cliftonii vermehren sich die Chloroplasten. Die Zunahme innerhalb von 2–3 Wochen betrug bis zu maximal 132%. Im Gegensatz dazu bleibt in nichtwachsenden, kernlosen Teilstücken aus der Basalregion der Zelle (Hinterstücke) die Chloroplastenzahl unverändert.Durch Verdunkelung in Gegenwart des Zellkernes (mit Rhizoid) läßt sich in Hinterstücken die Potenz für Wachstum induzieren, die nach Entfernung des Rhizoids im Licht realisiert werden kann. Gleichzeitig erhalten die Hinterstücke dabei die Fähigkeit zur Chloroplastenvermehrung.Wird in Hinterstücken die Chloroplastenzahl herabgesetzt (auf etwa ein Viertel des Normalwertes), so wird dadurch ebenfalls eine Vermehrung der Chloroplasten ausgelöst.Die Versuche sprechen dafür, daß die Vermehrung der Chloroplasten offenbar vom Verhältnis Chloroplastenprotein/Cytoplasmaprotein abhängig ist und eine Störung dieser Relation durch Abzentrifugieren der Chloroplasten bei diesen Teilungen auslöst. Die Korrelation zwischen Wachstum und Chloroplastenvermehrung dürfte auf ähnliche Weise zu erklären sein.

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Multiplication of chloroplasts in enucleated cell fragments ofAcetabularia mediterranea andAcetabularia cliftonii and its dependence from internal conditions

Summary Chloroplasts in growing enucleated cell fragments ofAcetabularia mediterranea andAcetabularia (Polyphysa) cliftonii multiply (obviously by division) and their number increases within 2–3 weeks up to 132%. However, there is no division of chloroplasts in non growing enucleated parts of the basal region of the cell.Capacity for growth can be induced in segments of the basal region which normally will not grow by dark treatment in presence of the nucleus. After removing the nucleus and transfering to light the enucleated parts grow and their number of chloroplasts increases.It is also possible to induce an increase of the number of chloroplasts by centrifugation of non growing enucleated cell fragments and thereby reducing the number of chloroplasts originally present.These results suggest that the increase in the number of chloroplasts very probably by division depends upon a certain ratio chloroplastic protein/cytoplasmic protein. Any disturbances of this ratio in favour of the cytoplasmic protein obviously leads to division of chloroplasts. It seems possible that the observed correlation between growth and the multiplication of chloroplast depends upon a similar mechanism.

     

Posttraumatic Stress Disorder (PTSD) Patients Exhibit a Blunted Parasympathetic Response to an Emotional Stressor

Diminished parasympathetic reactivity is a physiological feature of posttraumatic stress disorder (PTSD). The objective of this study was to compare female PTSD patients with non-traumatized healthy women with respect to autonomic cardiovascular regulation during exposure to two stressors. Hospitalized PTSD patients (n = 52) and controls (n = 37) completed standardized laboratory-based stress testing including a mental arithmetic test and a standardized audiotape recording of a crying infant. Controls and PTSD patients both showed a significantly increased heart rate and reduced pre-ejection period from baseline rest to the arithmetic stressor. However, as judged from nonsignificant changes in baroreflex sensitivity, parasympathetic activation caused by the crying infant stressor was blunted in PTSD patients as compared to healthy individuals. Under the crying infant condition, a vagal dominance was observed only in controls, and not in PTSD patients. Our data demonstrate that, in PTSD patients, diminished parasympathetic reactivity is not restricted to trauma-related events but can also be observed in response to a social stimulus such as listening to a crying infant. This finding suggests that the altered vagal reactivity in PTSD patients reflects the physiological consequences of a generally disturbed autonomous response to emotionally relevant stressors.     

Structure of the mosquitocidal toxin from Bacillus sphaericus

The catalytic domain of a mosquitocidal toxin prolonged by a C-terminal 44 residue linker connecting to four ricin B-like domains was crystallized. Three crystal structures were established at resolutions between 2.5A and 3.0A using multi-wavelength and single-wavelength anomalous X-ray diffraction as well as molecular replacement phasing techniques. The chainfold of the toxin fragment corresponds to those of ADP-ribosylating enzymes. At pH 4.3 the fragment is associated in a C(7)-symmetric heptamer in agreement with an aggregate of similar size observed by size-exclusion chromatography. In two distinct crystal forms, the heptamers formed nearly spherical, D(7)-symmetric tetradecamers. Another crystal form obtained at pH 6.3 contained a recurring C(2)-symmetric tetramer, which, however, was not stable in solution. On the basis of the common chainfold and NAD(+)-binding site of all ADP-ribosyl transferases, the NAD(+)-binding site of the toxin was assigned at a high confidence level. In all three crystal forms the NAD(+) site was occupied by part of the 44 residue linker, explaining the known inhibitory effect of this polypeptide region. The structure showed that the cleavage site for toxin activation is in a highly mobile loop that is exposed in the monomer. Since it contains the inhibitory linker as a crucial part of the association contact, the observed heptamer is inactive. Moreover, the heptamer cannot be activated by proteolysis because the activation loop is at the ring center and not accessible for proteases. Therefore the heptamer, or possibly the tetradecamer, seems to represent an inactive storage form of the toxin.     

A role for myelin-associated peroxisomes in maintaining paranodal loops and axonal integrity

Demyelinating diseases of the nervous system cause axon loss but the underlying mechanisms are not well understood. Here we show by confocal and electron microscopy that in myelin-forming glia peroxisomes are associated with myelin membranes. When peroxisome biogenesis is experimentally perturbed in Pex5 conditional mouse mutants, myelination by Schwann cells appears initially normal. However, in nerves of older mice paranodal loops become physically unstable and develop swellings filled with vesicles and electron-dense material. This novel model of a demyelinating neuropathy demonstrates that peroxisomes serve an important function in the peripheral myelin compartment, required for long-term axonal integrity.     

Social Immunity in Amphibians: Evidence for Vertical Transmission of Innate Defenses

Amphibian embryos are at risk of microbial infection. Here we find evidence that innate immune defenses, both antimicrobial skin peptides and mutualistic microbiota, of adult glass frogs, Hyalinobatrachium colymbiphyllum, can be transmitted to embryos deposited on leaves above rain forest streams in Panama and can inhibit the fungal pathogen Batrachochytrium dendrobatidis. Abstract in Spanish is available at http://www.blackwell‐synergy.com/loi/btp .