Secretion of biologically active glycoforms of bovine follicle stimulating hormone in plants.

We chose the follicle stimulating hormone (FSH), a pituitary heterodimeric glycoprotein hormone, as a model to assess the ability of the plant cell to express a recombinant protein that requires extensive N-glycosylation for subunit folding and assembly, intracellular trafficking, signal transduction and circulatory stability. A tobacco mosaic virus (TMV) based transient expression system was used to express a single-chain (sc) version of bovine FSH in the tobacco related species Nicotiana benthamiana. Preparations of periplasmic proteins from plants infected with recombinant viral RNA contained high levels of sc-bFSH, up to 3% of total soluble proteins. Consistently, in situ indirect immunofluorescence revealed that the plant cell secreted the mammalian secretory protein to the extracellular compartment (EC). By mass spectrometric analysis of immunoaffinity purified sc-bFSH derived from EC fractions, we found two species of the plant paucimannosidic glycan type, truncated forms of complex-type N-glycans. Stimulation of cAMP production in a CHO cell line expressing the porcine FSH receptor acknowledged the native-like structure of sc-bFSH and a sufficient extent of N-glycosylation required for signal transduction. Furthermore, in superovulatory treatments of mice, sc-bFSH displayed significant in vivo bioactivity, although much lower than that of pregnant mare serum gonadotropin. We conclude that plants may have a broad utility as hosts for the recombinant expression of proteins even where glycosylation is essential for function.     

Stimulatory pathways of the Calcium-sensing receptor on acid secretion in freshly isolated human gastric glands.

Gastric acid secretion is not only stimulated via the classical known neuronal and hormonal pathways but also by the Ca(2+)-Sensing Receptor (CaSR) located at the basolateral membrane of the acid-secretory gastric parietal cell. Stimulation of CaSR with divalent cations or the potent agonist Gd(3+) leads to activation of the H(+)/K(+)-ATPase and subsequently to gastric acid secretion. Here we investigated the intracellular mechanism(s) mediating the effects of the CaSR on H(+)/K(+)-ATPase activity in freshly isolated human gastric glands. Inhibition of heterotrimeric G-proteins (G(i) and G(o)) with pertussis toxin during stimulation of the CaSR with Gd(3+) only partly reduced the observed stimulatory effect. A similar effect was observed with the PLC inhibitor U73122. The reduction of the H(+)/K(+)-ATPase activity measured after incubation of gastric glands with BAPTA-AM, a chelator of intracellular Ca(2+), showed that intracellular Ca(2+) plays an important role in the signalling cascade. TMB-8, a ER Ca(2+)store release inhibitor, prevented the stimulation of H(+)/K(+)-ATPase activity. Also verapamil, an inhibitor of L-type Ca(2+)-channels reduced stimulation suggesting that both the release of intracellular Ca(2+) from the ER as well as Ca(2+) influx into the cell are involved in CaSR-mediated H(+)/K(+)-ATPase activation. Chelerythrine, a general inhibitor of protein kinase C, and Go 6976 which selectively inhibits Ca(2+)-dependent PKC(alpha) and PKC(betaI)-isozymes completely abolished the stimulatory effect of Gd(3+). In contrast, Ro 31-8220, a selective inhibitor of the Ca(2+)-independent PKCepsilon and PKC-delta isoforms reduced the stimulatory effect of Gd(3+) only about 60 %. On the other hand, activation of PKC with DOG led to an activation of H(+)/K(+)-ATPase activity which was only about 60 % of the effect observed with Gd(3+). Incubation of the parietal cells with PD 098059 to inhibit ERK1/2 MAP-kinases showed a significant reduction of the Gd(3+) effect. Thus, in the human gastric parietal cell the CaSR is coupled to pertussis toxin sensitive heterotrimeric G-Proteins and requires calcium to enhance the activity of the proton-pump. PLC, ERK 1/2 MAP-kinases as well as Ca(2+) dependent and Ca(2+)-independent PKC isoforms are part of the down-stream signalling cascade.     

Structural Mimicry of Receptor Interaction by Antagonistic Interleukin-6 (IL-6) Antibodies

Interleukin 6 plays a key role in mediating inflammatory reactions in autoimmune diseases and cancer, where it is also involved in metastasis and tissue invasion. Neutralizing antibodies against IL-6 and its receptor have been approved for therapeutic intervention or are in advanced stages of clinical development. Here we describe the crystal structures of the complexes of IL-6 with two Fabs derived from conventional camelid antibodies that antagonize the interaction between the cytokine and its receptor. The x-ray structures of these complexes provide insights into the mechanism of neutralization by the two antibodies and explain the very high potency of one of the antibodies. It effectively competes for binding to the cytokine with IL-6 receptor (IL-6R) by using side chains of two CDR residues filling the site I cavities of IL-6, thus mimicking the interactions of Phe²²⁹ and Phe²⁷⁹ of IL-6R. In the first antibody, a HCDR3 tryptophan binds similarly to hot spot residue Phe²⁷⁹. Mutation of this HCDR3 Trp residue into any other residue except Tyr or Phe significantly weakens binding of the antibody to IL-6, as was also observed for IL-6R mutants of Phe²⁷⁹. In the second antibody, the side chain of HCDR3 valine ties into site I like IL-6R Phe²⁷⁹, whereas a LCDR1 tyrosine side chain occupies a second cavity within site I and mimics the interactions of IL-6R Phe²²⁹.     

Life cycle assessment of construction and renovation of sewer systems using a detailed inventory tool

Purpose

The objective was to provide comprehensive life cycle inventories for the construction and renovation of sewers. A detailed inventory was provided with multiple options of pipe materials, diameters and site-specific characteristics, and was embedded into the Excel^®-based tool SewerLCA. The tool allows for life cycle evaluation of different sewers. It was applied to determine the most important phases, processes, and related parameters involved in the construction and renovation of sewers from an environmental and economical perspective.

Methods

Comprehensive life cycle inventories (LCIs) for sewers construction and renovation were obtained by first identifying all processes involved after interviewing construction experts and reviewing sewer construction budgets from a Catalan company; and second transforming the processes into masses of materials and energy usage using construction databases. In order to run the life cycle impact assessment (LCIA) the materials and energy typologies from the inventories were matched to their corresponding equivalents into available LCI databases. Afterwards the potential impacts were calculated through the use of LCIA characterization factors from ReCiPe. Life cycle assessment (LCA) was run several times to assess the construction of a 1-km-long sewer with varying pipe materials, life spans for each material, diameters, transport distances, site-specific characteristics, and pipe deposition options.

Results and discussion

The environmental impacts generated by construction and renovation of a 1 km Polyvinylchloride (PVC) pipe with a diameter of 40 cm are mainly associated with pipe laying and backfilling of the trench. The evaluation of several pipe materials and diameters shows that the exclusion of renovation would underestimate the impacts by 38 to 82 % depending on the pipe materials and diameters. Including end-of-life phase for plastic pipe materials increases climate change (up to an extra 71 %) and human toxicity (up to an extra 147 %) impacts (among all diameters). The preferred pipe materials from an environmental point of view are precast concrete and High-Density Polyethylene (HDPE). Site-specific characteristics (specially the presence of rocky soil and asphalt placement) and material life span have a high influence on the overall environmental profile, whereas changes in transport distances have only a minor impact (<4 %).

Conclusions

Environmental impacts during the construction and renovation of sewers are subject to differences in material type, site-specific characteristics and material life span. Renovation of sewers has a large influence on all potential environmental impacts and costs and, hence, should not be omitted in LCA studies. The treatment and disposal processes of plastic pipes at the end of their life has to be accounted in LCA studies.

     

Advances in transgenic animal models and techniques

On May 11th and 12th 2017 was held in Nantes, France, the international meeting “Advances in transgenic animal models and techniques” (http://www.trm.univ-nantes.fr/). This biennial meeting is the fifth one of its kind to be organized by the Transgenic Rats ImmunoPhenomic (TRIP) Nantes facility (http://www.tgr.nantes.inserm.fr/). The meeting was supported by private companies (SONIDEL, Scionics computer innovation, New England Biolabs, MERCK, genOway, Journal Disease Models and Mechanisms) and by public institutions (International Society for Transgenic Technology, University of Nantes, INSERM UMR 1064, SFR François Bonamy, CNRS, Région Pays de la Loire, Biogenouest, TEFOR infrastructure, ITUN, IHU-CESTI and DHU-Oncogeffe and Labex IGO). Around 100 participants, from France but also from different European countries, Japan and USA, attended the meeting.     

Spindle assembly defects leading to the formation of a monopolar mitotic apparatus

Mitotic spindle formation in animal cells involves microtubule nucleation from two centrosomes that are positioned at opposite sides of the nucleus. Microtubules are captured by the kinetochores and stabilized. In addition, microtubules can be nucleated independently of the centrosome and stabilized by a gradient of Ran—GTP, surrounding the mitotic chromatin. Complex regulation ensures the formation of a bipolar apparatus, involving motor proteins and controlled polymerization and depolymerization of microtubule ends. The bipolar apparatus is, in turn, responsible for faithful chromosome segregation. During recent years, a variety of experiments has indicated that defects in specific motor proteins, centrosome proteins, kinases and other proteins can induce the assembly of aberrant spindles with a monopolar morphology or with poorly separated poles. Induction of monopolar spindles may be a useful strategy for cancer therapy, since ensuing aberrant mitotic exit will usually lead to cell death. In this review, we will discuss the various underlying molecular mechanisms that may be responsible for monopolar spindle formation.