Bifunctional glycosyltransferases catalyze both extension and termination of pectic galactan oligosaccharides

Pectins are the most complex polysaccharides of the plant cell wall. Based on the number of methylations, acetylations and glycosidic linkages present in their structures, it is estimated that up to 67 transferase activities are involved in pectin biosynthesis. Pectic galactans constitute a major part of pectin in the form of side‐chains of rhamnogalacturonan‐I. In Arabidopsis, galactan synthase 1 (GALS1) catalyzes the addition of galactose units from UDP‐Gal to growing β‐1,4‐galactan chains. However, the mechanisms for obtaining varying degrees of polymerization remain poorly understood. In this study, we show that AtGALS1 is bifunctional, catalyzing both the transfer of galactose from UDP‐α‐d ‐Gal and the transfer of an arabinopyranose from UDP‐β‐l ‐Ara_p to galactan chains. The two substrates share a similar structure, but UDP‐α‐d ‐Gal is the preferred substrate, with a 10‐fold higher affinity. Transfer of Ara_p to galactan prevents further addition of galactose residues, resulting in a lower degree of polymerization. We show that this dual activity occurs both in vitro and in vivo. The herein described bifunctionality of AtGALS1 may suggest that plants can produce the incredible structural diversity of polysaccharides without a dedicated glycosyltransferase for each glycosidic linkage.     

Activation of PKCzeta and PKMzeta in the nucleus accumbens core is necessary for the retrieval, consolidation and reconsolidation of drug memory

One of the greatest challenges in the treatment of substance dependence is to reverse the control that drug-associated stimuli have gained over the addict's behavior, as these drug-associated memories increase the risk of relapse even after long periods of abstinence. We report here that inhibition of the atypical protein kinase C isoform PKCzeta and its constitutively active isoform PKMzeta with the pseudosubstrate inhibitor ZIP administered locally into the nucleus accumbens core reversibly inhibited the retrieval of drug-associated memory and drug (remifentanil) seeking, whereas a scrambled ZIP peptide or staurosporine, an effective inhibitor of c/nPKC-, CaMKII-, and PKA kinases that does not affect PKCzeta/PKMzeta activity, was without effect on these memory processes. Acquisition or extinction of drug-associated memory remained unaffected by PKCzeta- and PKMzeta inhibition.     

SpecNet: A Spatial Network Algorithm that Generates a Wide Range of Specific Structures

Network measures are used to predict the behavior of different systems. To be able to investigate how various structures behave and interact we need a wide range of theoretical networks to explore. Both spatial and non-spatial methods exist for generating networks but they are limited in the ability of producing wide range of network structures. We extend an earlier version of a spatial spectral network algorithm to generate a large variety of networks across almost all the theoretical spectra of the following network measures: average clustering coefficient, degree assortativity, fragmentation index, and mean degree. We compare this extended spatial spectral network-generating algorithm with a non-spatial algorithm regarding their ability to create networks with different structures and network measures. The spatial spectral network-generating algorithm can generate networks over a much broader scale than the non-spatial and other known network algorithms. To exemplify the ability to regenerate real networks, we regenerate networks with structures similar to two real Swedish swine transport networks. Results show that the spatial algorithm is an appropriate model with correlation coefficients at 0.99. This novel algorithm can even create negative assortativity and managed to achieve assortativity values that spans over almost the entire theoretical range.     

Synthesis of linear and cyclic phosphopeptides as ligands for the N-terminal SH2-domain of protein tyrosine phosphatase SHP-1.

Linear and cyclic phosphopeptides related to the pY2267 binding site of the epithelial receptor tyrosine kinase Ros have been synthesized as ligands for the amino-terminal SH2 (src homology) domain of protein tyrosine phosphatase SHP-1. The synthesis was accomplished by Fmoc-based solid-phase methodology using side-chain unprotected phosphotyrosine for the linear and mono-benzyl protected phosphotyrosine for the cyclic peptides. According to molecular modelling, the incorporation of a glycine residue between Lys (position pY-1 relative to phosphotyrosine) and Asp or Glu (position pY+2) was recommended for the cyclic candidates. The preparation of these peptides was successfully performed by the incorporation of a Fmoc-Xxx(Gly-OAll)-OH (Xxx = Asp, Glu) dipeptide building block that was prepared in solution prior to SPPS. The cyclization was achieved with PyBOP following Alloc/OAll-deprotection. This study demonstrates the usefulness of allyl-type protecting groups for the generation of side-chain cyclized phosphopeptides. Alloc/OAll-deprotection and cyclization are compatible with phosphorylated tyrosine.     

The Nucleotide-binding Leucine-rich Repeat (NLR) Family Member NLRX1 Mediates Protection against Experimental Autoimmune Encephalomyelitis and Represses Macrophage/Microglia-induced Inflammation

The nucleotide binding domain and leucine-rich repeat-containing (NLR) family of proteins is known to activate innate immunity, and the inflammasome-associated NLRs are prime examples. In contrast, the concept that NLRs can inhibit innate immunity is still debated, and the impact of such inhibitory NLRs in diseases shaped by adaptive immune responses is entirely unexplored. This study demonstrates that, in contrast to other NLRs that activate immunity, NLRX1 plays a protective role in experimental autoimmune encephalomyelitis (EAE), a mouse model for multiple sclerosis. When compared with wild-type controls, Nlrx1^−/− mice have significantly worsened clinical scores and heightened CNS tissue damage during EAE. NLRX1 does not alter the production of encephalitogenic T cells in the peripheral lymphatic tissue, but Nlrx1^−/− mice are more susceptible to adoptively transferred myelin-reactive T cells. Analysis of the macrophage and microglial populations indicates that NLRX1 reduces activation during both active and passive EAE models. This work represents the first case of an NLR that attenuates microglia inflammatory activities and protects against a neurodegenerative disease model caused by autoreactive T cells.     

The hedgehog receptor patched is involved in cholesterol transport

Background

Sonic hedgehog (Shh) signaling plays a crucial role in growth and patterning during embryonic development, and also in stem cell maintenance and tissue regeneration in adults. Aberrant Shh pathway activation is involved in the development of many tumors, and one of the most affected Shh signaling steps found in these tumors is the regulation of the signaling receptor Smoothened by the Shh receptor Patched. In the present work, we investigated Patched activity and the mechanism by which Patched inhibits Smoothened.

Methodology/Principal Findings

Using the well-known Shh-responding cell line of mouse fibroblasts NIH 3T3, we first observed that enhancement of the intracellular cholesterol concentration induces Smoothened enrichment in the plasma membrane, which is a crucial step for the signaling activation. We found that binding of Shh protein to its receptor Patched, which involves Patched internalization, increases the intracellular concentration of cholesterol and decreases the efflux of a fluorescent cholesterol derivative (BODIPY-cholesterol) from these cells. Treatment of fibroblasts with cyclopamine, an antagonist of Shh signaling, inhibits Patched expression and reduces BODIPY-cholesterol efflux, while treatment with the Shh pathway agonist SAG enhances Patched protein expression and BODIPY-cholesterol efflux. We also show that over-expression of human Patched in the yeast S. cerevisiae results in a significant boost of BODIPY-cholesterol efflux. Furthermore, we demonstrate that purified Patched binds to cholesterol, and that the interaction of Shh with Patched inhibits the binding of Patched to cholesterol.

Conclusion/Significance

Our results suggest that Patched may contribute to cholesterol efflux from cells, and to modulation of the intracellular cholesterol concentration. This activity is likely responsible for the inhibition of the enrichment of Smoothened in the plasma membrane, which is an important step in Shh pathway activation.     

Continuous hybridoma culture in a low-protein serum-free medium supplemented with liposomes

A homemade serum-free medium containing a low protein level under 0.1 g l⁻¹ has been proved to support long-term cultures of VO 208 hybridoma cells successfully up to 50 days. The low protein level was achieved by supplying the lipids through liposomes containing cholesterol, oleic acid, - dipalmitoyl phosphatidylcholine, and bovine serum albumin. The influence of the liposome content in the feeding medium was studied in a continuous culture performed with step variations of the liposomes level, from 7.5 to 30 ml l⁻¹. The cell density decreased at the highest liposomes content while it became higher with 7.5 or 12 ml l⁻¹ of liposomes. For each step variation appeared a transitory activation of the specific rates of nutrient consumption, metabolite production and antibody secretion, as well as a transitory decrease of the specific cell growth rate. The overall structure of the antibodies was not affected during the culture.     

Territorialit?t und Brutbiologie der FeldlercheAlauda arvensis in einer intensiv genutzten Agrarlandschaft

Zusammenfassung In einer intensiv landwirtschaftlich genutzten Fläche des schweizerischen Mittellandes wurden 1983–1987 vier Feldlerchenaggregationen mit maximal 39 Brutpaaren untersucht. Die Tradition (Ortstreue) bestimmte in erster Linie die Revierwahl und das Verteilungsmuster. Die Reviere waren mit ha sehr groß; ihre Größe korrelierte negativ mit der Anzahl Parzellen pro Fläche und der Kulturendiversität. Die Nutzung des Ressourcenangebots (Nistplatz, Nahrung, Schutz) war abhängig von den Fortbewegungsmöglichkeiten. In großparzellig strukturierten Flächen mit geringer Kulturendiversität kam es häufig zu Revierverschiebungen und Reviervergrößerungen. Die Feldlerche versucht, im Laufe der Brutperiode den Getreideanteil im Revier zu verkleinern und Kulturen wie Rüben, Kartoffeln und auch Mais zu aquirieren. Die Nistplatzwahl zeigte eine deutliche Präferenz für alle nicht zu dicht stehenden, grasartigen Kulturen (Weizen, Hafer, Fettwiese). Eine Vegetationshöhe von 15–25 cm und eine Bodenbedeckung von 20–50 % bieten optimale Bedingungen für den Nestbau. 51 % aller Nester wurden in der Fettwiese angelegt, 20 % im Winterweizen und 18 % im Mais. Je nach Bewirtschaftungsart brüteten die im Mittel 2,4–2,8mal pro Brutsaison. Die Schlüpfrate von 220 Gelegen betrug 0,58. 44 % der geschlüpften Jungvögel verließen das Nest. Der Bruterfolg (nestverlassende Junge/gelegte Eier) lag bei max. 25 %. Dem Fütterungsverhalten der Altvögel nach zu schließen, wurden maximal 50 % der nestverlassenden Jungen flügge. Durchschnittlich war mit 0,9 flüggen Jungen pro Brutpaar und Jahr zu rechnen. Brutverluste waren kulturspezifisch und daher in erster Linie vom Neststandort abhängig.

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Territoriality and breeding biology of Skylark (Alauda arvensis) in an intensively farmed area in Switzerland

Summary In 1983–1987 four plots (total surface 307 ha) with up to 39 breeding pairs were studied in an area of intensive cultivation in the Swiss Lowlands (390 m asl). Distribution pattern and selection of territories are determined by a strong site tenacity of the birds. The size of territories averaged 3,3±0,9 hectares. It correlates negatively with the number of different crops per unit area and the diversity of cultures.The mobility within the vegetation determined which types of farmland ressources may be used for nesting, feeding and cover. Shifts of territories and attempts to enlarge them were frequent in areas of large plots with low crop diversity. During the season, Skylarks tried to reduce the proportion of cereal fields within their territory and to gain plots planted with sugar-beet, potatoes and corn.Their choice of nesting sites showed a strong preference for less dense grass crops such as wheat, oat or meadows. Optimal conditions for nest sites were in fields with a vegetation height of 15–25 cm and a ground coverage of 20–50 %. 51 % of 220 nests were constructed in heavily manured meadows, 20 % in winter wheat fields and 18 % in corn fields. Females bred on average 2,4–2,8 times per breeding season, depending on the type of cultivation (agriculture, husbandry). Hatching rate was 58 % out of 220 clutches, and 44 % of the hatched young left the nest. Maximum breeding success (i. e. young birds leaving the nest/number of eggs laid) was thus maximal 25 %. Observations of parental feeding behaviour suggest that a maximum of 50 % of the young larks which leave the nests survive to the age of fledging. It is estimated that 0,9 fledglings are raised per breeding pairs and year. Losses of broods vary according to farming practises in the different crops and therefore are largely depending on nesting sites.

     

Characterization of NLRP12 during the development of allergic airway disease in mice

Among the 22 members of the nucleotide binding-domain, leucine rich repeat-containing (NLR) family, less than half have been functionally characterized. Of those that have been well studied, most form caspase-1 activating inflammasomes. NLRP12 is a unique NLR that has been shown to attenuate inflammatory pathways in biochemical assays and mediate the lymph node homing of activated skin dendritic cells in contact hypersensitivity responses. Since the mechanism between these two important observations remains elusive, we further evaluated the contribution of NLRP12 to organ specific adaptive immune responses by focusing on the lung, which, like skin, is exposed to both exogenous and endogenous inflammatory agents. In models of allergic airway inflammation induced by either acute ovalbumin (OVA) exposure or chronic house dust mite (HDM) antigen exposure, Nlrp12(-/-) mice displayed subtle differences in eosinophil and monocyte infiltration into the airways. However, the overall development of allergic airway disease and airway function was not significantly altered by NLRP12 deficiency. Together, the combined data suggest that NLRP12 does not play a vital role in regulating Th2 driven airway inflammation using common model systems that are physiologically relevant to human disease. Thus, the allergic airway inflammation models described here should be appropriate for subsequent studies that seek to decipher the contribution of NLRP12 in mediating the host response to agents associated with asthma exacerbation.