Development of a S. cerevisiae whole cell biocatalyst for in vitro sialylation of oligosaccharides

Absence of sialylation on recombinant glycoproteins compromises their efficacy as therapeutic agents, as it results in rapid clearance from the human bloodstream. To circumvent this, several strategies are followed, including the implementation of a post-secretion glycosylation step. In this paper we describe the engineering of yeast cells expressing active surface exposed Trypanosoma cruzi trans-sialidase (TS) fused to the yeast Aga2 protein, and the use of this yeast in the sialylation of synthetic oligosaccharides. In an attempt to improve overall protein accessibility on the yeast surface, we abolished hyperglycosylation on the yeast cell wall proteins. This was achieved by disrupting the OCH1 gene of the TS surface expressing strain, which resulted in increased enzymatic activity. Using a fluorescence-based activity assay and DSA-FACE structural analysis, we obtained almost complete conversion to a fully sialylated acceptor, whereas in the wild type situation this conversion was only partial. Increasing protein accessibility on the yeast surface by modifying the glycosylation content thus proved to be a valuable approach in increasing the cell wall associated activity of an immobilised enzyme, hence resulting in a more effective biocatalyst system.     

Waste Treatment and Assessment of Long-Term Emissions (8pp)

Goal, Scope and Background The disposal phase of a product’s life cycle in LCA is often neglected or based on coarse indicators like ‘kilogram waste’. The goal of report No. 13 of the ecoinvent project (Doka 2003) is to create detailed Life Cycle Inventories of waste disposal processes. The purpose of this paper is to give an overview of the models behind the waste disposal inventories in ecoinvent, to present exemplary results and to discuss the assessment of long-term emissions. This paper does not present a particular LCA study. Inventories are compiled for many different materials and various disposal technologies. Considered disposal technologies are municipal incineration and different landfill types, including sanitary landfills, hazardous waste incineration, waste deposits in deep salt mines, surface spreading of sludges, municipal wastewater treatment, and building dismantling. The inventoried technologies are largely based on Swiss plants. Inventories can be used for assessment of the disposal of common, generic waste materials like paper, plastics, packaging etc. Inventories are also used within the ecoinvent database itself to inventory the disposal of specific wastes generated during the production phase. Inventories relate as far as possible to the specific chemical composition of the waste material (waste-specific burdens). Certain expenditures are not related to the waste composition and are inventoried with average values (process-specific burdens). Methods The disposal models are based on previous work, partly used in earlier versions of ecoinvent/ETH LCI data. Important improvements were the extension of the number of considered chemical elements to 41 throughout all disposal models and new landfill models based on field data. New inventories are compiled for waste deposits in deep salt mines and building material disposal. Along with the ecoinvent data and the reports, also Excel-based software tools were created, which allow ecoinvent members to calculate waste disposal inventories from arbitrary waste compositions. The modelling of long-term emissions from landfills is a crucial part in any waste disposal process. In ecoinvent long-term emissions are defined as emissions occurring 100 years after present. They are reported in separate emission categories. The landfill inventories include long-term emissions with a time horizon of 60’000 years after present. Results and Discussion As in earlier studies, the landfills prove to be generally relevant disposal processes, as also incineration and wastewater treatment processes produce landfilled wastes. Heavy metals tend to concentrate in landfills and are washed out to a varying degree over time. Long-term emissions usually represent an important burden from landfills. Comparisons between burdens from production of materials and the burdens from their disposal show that disposal has a certain relevance. Conclusion The disposal phase should by default be included in LCA studies. The use of a material not only necessitates its production, but also requires its disposal. The created inventories and user tools facilitate heeding the disposal phase with a similar level of detail as production processes. The risk of LCA-based decisions shifting burdens from the production or use phase to the disposal phase because of data gaps can therefore be diminished. Recommendation and Perspective Future improvements should include the modelling of metal ore refining waste (tailings) which is currently neglected in ecoinvent, but is likely to be relevant for metals production. The disposal technologies considered here are those of developed Western countries. Disposal in other parts of the World can differ distinctly, for logistic, climatic and economic reasons. The cross-examination of landfill models to LCIA soil fate models could be advantageous. Currently only chemical elements, like copper, zinc, nitrogen etc. are heeded by the disposal models. A possible extension could be the modelling of the behaviour of chemical compounds, like dioxins or other hydrocarbons.     

Antigen-driven interactions with dendritic cells and expansion of Foxp3+ regulatory T cells occur in the absence of inflammatory signals

Foxp3+ regulatory T cells (Tregs) play a pivotal role in the maintenance of peripheral T cell tolerance and are thought to interact with dendritic cells (DC) in secondary lymphoid organs. We analyzed here the in vivo requirements for selective expansion of Ag-specific Treg vs CD4+CD25- effector T cells and engagement of Ag-specific Treg-DC interactions in secondary lymphoid organs. Using i.v. Ag delivery in the absence of inflammation, we found that CD4+CD25+Foxp3+ Tregs undergo vigorous expansion and accumulate whereas naive CD4+CD25-Foxp3- T cells undergo abortive activation. Quantifying directly the interactions between Tregs and CD11c+ DC, we found that Tregs establish cognate contacts with endogenous CD11c+ DC in spleen and lymph nodes at an early time point preceding their expansion. Importantly, we observed that as few as 10(3) Tregs selectively expanded by i.v. Ag injection are able to suppress B and T cell immune responses in mouse recipients challenged with the Ag. Our results demonstrate that Tregs are selectively mobilized by Ag recognition in the absence of inflammatory signals, and can induce thereafter potent tolerance to defined Ag targets.     

Synthetic substrate analogues for UDP-GlcNAc: Manα1-6R β(1-2)-N-acetylglucosaminyltransferase II. Substrate specificity and inhibitors for the enzyme

UDP-GlcNAc: Man1-6R (1-2)-N-acetylglucosaminyltransferase II (GlcNAc-T II; EC 2.4.1.143) is a key enzyme in the synthesis of complexN-glycans. We have tested a series of synthetic analogues of the substrate Man1-6(GlcNAc1-2Man1-3)Man-O-octyl as substrates and inhibitors for rat liver GlcNAc-T II. The enzyme attachesN-acetylglucosamine in 1-2 linkage to the 2-OH of the Man1-6 residue. The 2-deoxy analogue is a competitive inhibitor (K _i=0.13mm). The 2-O-methyl compound does not bind to the enzyme presumably due to steric hindrance. The 3-, 4- and 6-OH groups are not essential for binding or catalysis since the 3-, 4- and 6-deoxy and -O-methyl derivatives are all good substrates. Increasing the size of the substituent at the 3-position to pentyl and substituted pentyl groups causes competitive inhibition (K _i=1.0–2.5mm). We have taken advantage of this effect to synthesize two potentially irreversible GlcNAc-T II inhibitors containing a photolabile 3-O-(4,4-azo)pentyl group and a 3-O-(5-iodoacetamido)pentyl group respectively. The data indicate that none of the hydroxyls of the Man1-6 residue are essential for binding although the 2- and 3-OH face the catalytic site of the enzyme. The 4-OH group of the Man-O-octyl residue is not essential for binding or catalysis since the 4-deoxy derivative is a good substrate; the 4-O-methyl derivative does not bind. This contrasts with GlcNAc-T I which cannot bind to the 4-deoxy-Man- substrate analogue. The data are compatible with our previous observations that a bisectingN-acetylglucosamine at the 4-OH position prevents both GlcNAc-T I and GlcNAc-T II catalysis. However, in the case of GlcNAc-T II, the bisectingN-acetylglucosamine prevents binding due to steric hindrance rather than to removal of an essential OH group. The 3-OH of the Man1-3 is an essential group for GlcNAc-T II since the 3-deoxy derivative does not bind to the enzyme. The trisaccharide GlcNAc1-2Man1-3Man-O-octyl is a good inhibitor (K _i=0.9mm). The above data together with previous studies indicate that binding of the GlcNAc1-2Man1-3Man- arm of the branched substrate to the enzyme is essential for catalysis. Abbreviations: GlcNAc-T I, UDP-GlcNAc:Man1-3R (1-2)-N-acetylglucosaminyltransferase I (EC 2.4.1.101); GlcNAc-T II, UDP-GlcNAc:Man1-6R (1-2)-N-acetylglucosaminyltransferase II (EC 2.4.1.143); MES, 2-(N-morpholino)ethane sulfonic acid monohydrate.     

Synthesis of Brunfelsamidine Ribonucleoside and Certain Related Compounds by the Stereospecific Sodium Salt Glycosylation Procedure

Abstract

A synthesis of 2,4-dideazaribavirin ( 2 ), brunfelsamidine ribonucleoside ( 8c ) and certain related derivatives are described for the first time using the stereospecific sodium salt glycosylation procedure. Glycosylation of the sodium salt of pyrrole-3-carbonitrile ( 4 ) with 1-chloro-2, 3-O-t-isopropylidene-5-O-t-butyldimethylsilyl-α-D-ribofuranose ( 5 ) gave exclusively the corresponding blocked nucleoside ( 6 ) with β-anomeric configuration, which on deprotection provided 1-β-D-ribofuranosylpyrrole-3-carbonitrile ( 7 ). Functional group tranformation of 7 gave 2 , 8c and related 3-substituted pyrrole ribonucleosides. These compounds are devoid of any significant antiviral/antitumor activity invitro.     

Circadian Pattern of Wheel‐Running Activity of a South American Subterranean Rodent (Ctenomys cf knightii)

Circadian rhythms are regarded as essentially ubiquitous features of animal behavior and are thought to confer important adaptive advantages. However, although circadian systems of rodents have been among the most extensively studied, most comparative biology is restricted to a few related species. In this study, the circadian organization of locomotor activity was studied in the subterranean, solitary north Argentinean rodent, Ctenomys knightii. The genus, Ctenomys, commonly known as Tuco‐tucos, comprises more than 50 known species over a range that extends from 12°S latitude into Patagonia, and includes at least one social species. The genus, therefore, is ideal for comparative and ecological studies of circadian rhythms. Ctenomys knightii is the first of these to be studied for its circadian behavior. All animals were wild caught but adapted quickly to laboratory conditions, with clear and precise activity‐rest rhythms in a light‐dark (LD) cycle and strongly nocturnal wheel running behavior. In constant dark (DD), the rhythm expression persisted with free‐running periods always longer than 24 h. Upon reinstatement of the LD cycle, rhythms resynchronized rapidly with large phase advances in 7/8 animals. In constant light (LL), six animals had free‐running periods shorter than in DD, and 4/8 showed evidence of “splitting.” We conclude that under laboratory conditions, in wheel‐running cages, this species shows a clear nocturnal rhythmic organization controlled by an endogenous circadian oscillator that is entrained to 24 h LD cycles, predominantly by light‐induced advances, and shows the same interindividual variable responses to constant light as reported in other non‐subterranean species. These data are the first step toward understanding the chronobiology of the largest genus of subterranean rodents.     

The Caribou Migration Model for Arctic Hadrosaurs (Dinosauria: Ornithischia): A Reassessment

The recovery of abundant dinosaur fossils from high paleolatitudes of northern Alaska has raised some hard questions in relation to any available model of dinosaur physiology. To explain the occurrence of hadrosaurs at high ancient latitudes, a model involving long-distance migration analogous to that of the modern caribou has been proposed. The model calls for seasonal movements over great latitudinal distances by these Cretaceous hadrosaurs. This model is reassessed in terms of the growth, body sizes, and inferred physiological ecology of the hadrosaurs and the caribou. Histological data suggest that juvenile hadrosaurs obtained from northern Alaska were greater than 1 year in age. Comparison of the relative sizes of juvenile and adult hadrosaurs with juvenile and adult caribou suggests, based on qualitative energetics, that the juvenile hadrosaurs were too small to participate in long-distance migration. The "hadrosaurs as caribou" model provides clues to the feeding ecology of North Slope hadrosaurs, if they are reinterpreted as year-round residents of high latitudes. However, it does not constitute a satisfactory basis on which to infer long-distance seasonal migrations by these animals.     

Cardiopulmonary bypass reduces atrial Na+-K+-ATPase expression in children

Cardiopulmonary bypass (CPB) may induce serious side effects, potentially leading to myocardial failure. The Na(+)-K(+)-ATPase is a key component for myocardial function. Due to its developmental regulation, results from adult studies cannot be adopted to the situation in childhood. Right atrial myocardium from patients with left-to-right shunts at atrial level (VO, n=8) and those without (NO, n=8) was excised during heart surgery before and after CPB. Na(+)-K(+)-ATPase isoforms ATP1A1 (p=0.008) and ATP1A3 (p=0.038) decreased during CPB, which decrease was restricted to the VO group. This study highlights the importance of the underlying heart defect for susceptibility to the effects of CPB, showing a reduced Na(+)-K(+)-ATPase mRNA expression only in patients with left-to-right shunts on the atrial level. This seemed to be an early molecular event, as apart from one, none of the patients showed heart failure before or after surgery.     

Anthrax toxin protective antigen: inhibition of channel function by chloroquine and related compounds and study of binding kinetics using the current noise analysis

Protective antigen (PA) of the tripartite anthrax toxin binds to a cell surface receptor and mediates the transport of two enzymatic components, edema factor and lethal factor, into the cytosol of host cells. Here recombinant PA(63) from Bacillus anthracis was reconstituted into artificial lipid bilayer membranes and formed ion permeable channels. The heptameric PA(63)-channel contains a binding site for 4-aminoquinolones, which block ion transport through PA in vitro. This result allowed a detailed investigation of ligand binding and the stability constants for the binding of chloroquine, fluphenazine, and quinacrine to the binding site inside the PA(63)-channel were determined using titration experiments. Open PA(63)-channels exhibit 1/f noise in the frequency range between 1 and 100 Hz, whereas the spectral density of the ligand-induced current noise was of Lorentzian type. The analysis of the power density spectra allowed the evaluation of the on- and off-rate constants (k(1) and k(-1)) of ligand binding. The on-rate constants of ligand binding were between 10(6) and 10(8) M(-1) s(-1) and were dependent on the ionic strength of the aqueous phase, sidedness of ligand addition, as well as the orientation and intensity of the applied electric field. The off-rates varied between approximately 10 s(-1) and 2600 s(-1) and depended mainly on the structure of the ligand.     

Hypotonic stress induces E-cadherin expression in cultured human keratinocytes

Human epidermis marks the interface between internal and external environments with the major task being to maintain body hydration. Alternating exposure of skin to a dry or humid environment is likely to cause changes in the epidermal water gradient resulting in osmotic alterations of epidermal keratinocytes. The present in vitro approach studied the effect of hypotonicity on cell-cell contact. It was demonstrated that hypotonic stress applied to human epithelial cells (HaCaT, A-431) induced upregulation of E-cadherin at both, the protein and mRNA level. 5'-deletional mutants of the E-cadherin promoter identified an element ranging from -53 to +31 that conveyed strong transactivation under hypotonic stress. In order to define relevant upstream regulators members of the MAP kinase family, the epidermal growth factor receptor (EGFR) and protein kinase B/Akt (PKB/Akt) were investigated. Hypotonic conditions led to a fast activation of ERK1/2, SAPK/JNK, p38, EGFR and PKB/Akt with distinct activation patterns. Experiments using specific inhibitors showed that p38 contributes to the E-cadherin transactivation under hypotonic conditions. Further upstream, adhesion was found to be a prerequisite for E-cadherin transactivation in this model. In summary, the present study provides evidence that E-cadherin is an osmo-sensitive gene that responds to hypotonic stress. The function of this regulation may be found in morphological changes induced by cell swelling. It is likely that induction of E-cadherin contributes to the stabilization between adjacent cells in order to withstand the physical forces induced by hypotonicity.