Coiled-Coil Formation on Lipid Bilayers—Implications for Docking and?Fusion Efficiency

Coiled-coil formation of four different oligopeptides was characterized in solution, on hydrogels, and on membranes by employing circular dichroism spectroscopy, surface plasmon resonance spectroscopy, attenuated total reflection infrared spectroscopy, and ellipsometry. Peptide sequences rich in either glutamic acid (E: E3Cys, i-E3Cys) or lysine (K: K3Cys, i-K3Cys) were used to represent minimal mimics of eukaryotic SNARE motifs. Half of the peptides were synthesized in reverse sequence, so that parallel and antiparallel heptad coiled-coil structures were formed. Either E-peptides or K-peptides were attached covalently to phospholipid anchors via maleimide chemistry, and served as receptors for the recognition of the corresponding binding partners added to solution. Attenuated total reflection infrared spectroscopy of single bilayers confirmed the formation of coiled-coil complexes at the membrane interface. Coiled-coil formation in solution, as compared with association at the membrane surface, displays considerably larger binding constants that are largely attributed to loss of translational entropy at the interface. Finally, the fusogenicity of the various coiled-coil motifs was explored, and the results provide clear evidence that hemifusion followed by full fusion requires a parallel orientation of α-helices, whereas antiparallel oriented coiled-coil motifs display only docking.     

Long-distance dispersal of a wolf, <Emphasis Type="Italic">Canis lupus</Emphasis>, in northwestern Europe

Several mammal species have recolonized their historical ranges across Europe during the last decades. In November 2012, a wolf-looking canid was found dead in Thy National Park (56° 56′ N, 8° 25′ E) in Jutland, Denmark. DNA from this individual and nine German wolves were genotyped using a genome-wide panel of 22,163 canine single nucleotide polymorphism (SNP) markers and compared to existing profiles based on the same marker panel obtained from northeastern Polish (n?=?13) wolves, domestic dogs (n?=?13) and known wolf-dog hybrids (n?=?4). The Thy canid was confirmed to be a wolf from the German-western Polish population, approximately 800 km to the southeast. Access to the German reference database on DNA profiles based on 13 autosomal microsatellites of German wolves made it possible to pinpoint the exact pack origin of the Thy wolf in Saxony, Germany. This was the first documented observation of a wolf in Denmark in 200 years and another example of long-distance dispersal of a carnivore.     

Differences between single and paired heifers in residency in functional areas, length of travel path, and area used throughout days 1–6 after integration into a free stall dairy herd

The integration of heifers into free stall dairy herds is a frequent management procedure, but little systematic research has been conducted on its effect on cow behavior. Previous studies mainly focused on aggressive interactions, but it is also of interest how integration affects the spatial distribution of both the cows in the herd and the integrated heifers. In the present study we integrated a single and a pair of heifers on each of six Swiss working farms in a balanced order. Using an automatic tracking system, we recorded the positions of all the cows and of the integrated heifers at 1 min intervals for six continuous 24 h periods. From these data we calculated the proportion of time the animals spent in the activity area, at the feed rack and in the lying cubicles, their average path length and the area of the barn that they used. We then compared the behavior of the integrated heifers with that of the cows in the introductory weeks. We also compared the behavior of the cows recorded in the control weeks directly preceding the integration and in the introductory weeks. For evaluation we used linear mixed-effects models. Singly integrated heifers spent a higher proportion of time in the activity area (0.29 vs. 0.14; P < 0.001) and a lower proportion of time in the lying area (0.40 vs. 0.53; interaction with day, P = 0.011) than the cows, whereas the heifers of the pairs mainly spent a lower proportion of time in the feeding area than the cows (0.23 vs. 0.32; interaction with day, P = 0.044). Average path length was longer for the integrated heifers soon after introduction but approached the values of the cows later on (interaction with day, P = 0.012). The total barn area used by a given animal was largest in the cows and was reduced in heifers integrated singly or in pairs (cows: 341/373 m², pairs: 306 m², single heifer: 333 m²; P = 0.055). Cows were little influenced in their space use by the integration of a single or pair of heifers. In summary, the behavior of the singly integrated heifers differed more markedly from that of the cows than the behavior of the heifers introduced in pairs during the introductory week. We would therefore recommend integrating pairs rather than single heifers into herds of dehorned dairy cows to ease their integration.     

Choice of scan-sampling intervals—An example with quantifying neighbours in dairy cows

To date, only a few studies using scan-sampling intervals to record neighbours in groups of animals explain how the interval length was chosen. In this study, we investigated the effects of different scan-sampling intervals, behavioural context, and the definition of neighbours on the accuracy of data as compared to a quasi-continuous observation. The data was collected from dairy cows kept in cubicle housing systems. Dairy herds of 22–43 cows on 6 farms were observed once a minute with an automatic tracking system for 6 days, and both the nearest neighbour and all neighbours within a defined distance of each cow were recorded with references to the activity, feeding and lying area and stored in neighbourship matrices. In the analysis, we used data collected at intervals of 2, 3, 4, …, 30 min, and correlated this simulated data with the data based on the 1-min interval using a specialised correlation coefficient for matrices τ_Kr. This correlation coefficient was then used as the response variable in mixed-effects models. We found that the size of the correlation coefficients generally decreased as interval length increased. This decrease was less pronounced for all neighbours than for the nearest neighbour. Moreover, the decrease was greatest with data from the activity area and lowest with data from the lying area. We concluded that, even with the relatively slow dairy cows in a barn environment, neighbour recordings should be conducted at short scan-sampling intervals in order to achieve a minimum correlation of τ_Kr = 0.8 with the quasi-continuous data. Intervals of every 2, 8 and 17 min are recommended for observation of neighbours in dairy cows for the activity, feeding and lying areas, respectively, and species that move faster may well require even shorter sampling intervals.     

Feeding ecology of wolvesCanis lupus returning to Germany

Following several years of occasional occurrence, several wolvesCanis lupus Linnaeus, 1758 have established a resident population in northeastern Saxony (Eastern Germany). From 2001 to 2003, we collected and analysed 192 scats ofC. lupus. Results of our study are expressed as the frequency of occurrence of prey species and the percentage of biomass consumed using coefficients of digestibility as well as two variants of an equation for prey mass per collectable scat. Diet composition of the wolves was restricted to a few food items, mostly wild ungulates. These remains were found in 97% of the scats, representing 99% of the biomass consumed by the wolves. Roe deerCapreolus capreolus was the most frequent and most important prey, constituting nearly of one half the biomass. Red deerCervus elaphus was recorded in one-third of the samples, followed by wild boarSus scrofa, mouflonOvis am mon musimon and brown hareLepus europaeus. Compared with game occurrence, roe deer was clearly preferred over the other species. A difference between winter and summer diets was mainly due to the high occurrence of young wild boar in summer. The general diet pattern of the wolf in Saxony corresponds with that found in the naturally occurring populations in Europe.     

The Phosphate Source Influences Gene Expression and Quality of Mineralization during In Vitro Osteogenic Differentiation of Human Mesenchymal Stem Cells

For in vitro differentiation of bone marrow-derived mesenchymal stem cells/mesenchymal stromal cells into osteoblasts by 2-dimensional cell culture a variety of protocols have been used and evaluated in the past. Especially the external phosphate source used to induce mineralization varies considerably both in respect to chemical composition and concentration. In light of the recent findings that inorganic phosphate directs gene expression of genes crucial for bone development, the need for a standardized phosphate source in in vitro differentiation becomes apparent. We show that chemical composition (inorganic versus organic phosphate origin) and concentration of phosphate supplementation exert a severe impact on the results of gene expression for the genes commonly used as markers for osteoblast formation as well as on the composition of the mineral formed. Specifically, the intensity of gene expression does not necessarily correlate with a high quality mineralized matrix. Our study demonstrates advantages of using inorganic phosphate instead of β-glycerophosphate and propose colorimetric quantification methods for calcium and phosphate ions as cost- and time-effective alternatives to X-ray diffraction and Fourier-transform infrared spectroscopy for determination of the calcium phosphate ratio and concentration of mineral matrix formed under in vitro-conditions. We critically discuss the different assays used to assess in vitro bone formation in respect to specificity and provide a detailed in vitro protocol that could help to avoid contradictory results due to variances in experimental design.     

Einige Feinstrukturbefunde an den Plexus chorioidei von Affen

Zusammenfassung An der lichtmikroskopischen Struktur des plexus chorioideus ventriculi III von Ateles, Cebus, Macaca und Pan fallen deutliche speziesbedingte Unterschiede auf. Bei Macaca wurde die Ultrastruktur der Plexus chorioidei ventriculi III und IV sowie der Plexus chorioidei der Seitenventrikel verglichen. Kuppenzellen überwiegen im elektronen-mikroskopischen Bild der Plexusepithelien des III. und IV. Ventrikels, während unter den Epithelien der Seitenventrikel flachere Zellen dominieren. Diese strukturellen Besonderheiten könnten auf funktionelle Unterschiede in der Liquorbildung hinweisen. Das Problem der extrachorioidalen Liquorproduktion (Curl and Pollay, 1968; Milhorat u. Mitarb., 1971) wird im Zusammenhang mit den regionalen Unterschieden in der Wandstruktur des Aquaeductus cerebri (Sylvii) diskutiert (Merker, 1970). Im Stroma und im Interstitium aller Plexus chorioidei des Rhesusaffen sind makrophagenartige Wanderzellen zu beobachten, deren Strukturbild dem der Epiplexuszellen gleicht; sie werden als ausgewanderte Monozyten gedeutet. Wahrscheinlich gelangen diese Zellen in den Ventrikel und werden dort zu Epiplexuszellen. Einschlüsse vom Typ der Biondi-Körper wurden in den Plexusepithelien der untersuchten Affen nicht beobachtet; die untersuchten Tiere hatten aber noch kein hohes Lebensalter erreicht.

---

The fine structure of the choroid plexuses in monkeys

Summary The light microscopic structure of the choroid plexus of the III^rd ventricle was studied in Ateles, Cebus, Macaca and Pan. These choroid plexuses display distinct species differences. The ultrastructure of the choroid plexuses of the III^rd, IV^th and lateral ventricles was compared in Macaca. Cuboidal cells with dome-like protrusions predominate in the epithelium of the choroid plexuses of the III^rd and the IV^th ventricles, while flattened cells are characteristic of the choroid epithelium of the lateral ventricle. These structural peculiarities may reflect functional differences in the production of the cerebrospinal fluid. The problem of extrachoroidal production of the cerebrospinal fluid (Curl and Pollay, 1968; Milhorat et al., 1971) is discussed in connection with regional differences in the structure of the aqueduct wall (Merker, 1970). Macrophage-like cells which morphologically resemble epiplexus cells and which are very abundant within the connective tissue stroma and also between the epithelial cells of all choroid plexuses of the rhesus monkey, are thought to be monocytes. Probably these cells migrate into the ventricle where they become epiplexus cells. Epithelial inclusions of the type of Biondi bodies were not observed in the simian choroid plexuses. However, the animals investigated were not old.

Mit Unterstützung durch die Deutsche Forschungsgemeinschaft (Arbeitskreis Prof. Dr. A. Oksche).     

Controls of dissolved organic matter quality: evidence from a large‐scale boreal lake survey

Inland waters transport large amounts of dissolved organic matter (DOM) from terrestrial environments to the oceans, but DOM also reacts en route, with substantial water column losses by mineralization and sedimentation. For DOM transformations along the aquatic continuum, lakes play an important role as they retain waters in the landscape allowing for more time to alter DOM. We know DOM losses are significant at the global scale, yet little is known about how the reactivity of DOM varies across landscapes and climates. DOM reactivity is inherently linked to its chemical composition. We used fluorescence spectroscopy to explore DOM quality from 560 lakes distributed across Sweden and encompassed a wide climatic gradient typical of the boreal ecozone. Six fluorescence components were identified using parallel factor analysis (PARAFAC). The intensity and relative abundance of these components were analyzed in relation to lake chemistry, catchment, and climate characteristics. Land cover, particularly the percentage of water in the catchment, was a primary factor explaining variability in PARAFAC components. Likewise, lake water retention time influenced DOM quality. These results suggest that processes occurring in upstream water bodies, in addition to the lake itself, have a dominant influence on DOM quality. PARAFAC components with longer emission wavelengths, or red‐shifted components, were most reactive. In contrast, protein‐like components were most persistent within lakes. Generalized characteristics of PARAFAC components based on emission wavelength could ease future interpretation of fluorescence spectra. An important secondary influence on DOM quality was mean annual temperature, which ranged between ?6.2 and +7.5 °C. These results suggest that DOM reactivity depends more heavily on the duration of time taken to pass through the landscape, rather than temperature. Projected increases in runoff in the boreal region may force lake DOM toward a higher overall amount and proportion of humic‐like substances.