Trypanosoma brucei UDP-Glucose:Glycoprotein Glucosyltransferase Has Unusual Substrate Specificity and Protects the Parasite from Stress

In this paper, we describe the range of N-linked glycan structures produced by wild-type and glucosidase II null mutant bloodstream form Trypanosoma brucei parasites and the creation and characterization of a bloodstream form Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase null mutant. These analyses highlight peculiarities of the Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase, including an unusually wide substrate specificity, ranging from Man₅GlcNAc₂ to Man₉GlcNAc₂ glycans, and an unusually high efficiency in vivo, quantitatively glucosylating the Asn263 N-glycan of variant surface glycoprotein (VSG) 221 and 75% of all non-VSG N glycosylation sites. We also show that although Trypanosoma brucei UDP-glucose:glycoprotein glucosyltransferase is not essential for parasite growth at 37°C, it is essential for parasite growth and survival at 40°C. The null mutant was also shown to be hypersensitive to the effects of the N glycosylation inhibitor tunicamycin. Further analysis of bloodstream form Trypanosoma brucei under normal conditions and stress conditions suggests that it does not have a classical unfolded protein response triggered by sensing unfolded proteins in the endoplasmic reticulum. Rather, judging by its uniform Grp78/BiP levels, it appears to have an unregulated and constitutively active endoplasmic reticulum protein folding system. We suggest that the latter may be particularly appropriate for this organism, which has an extremely high flux of glycoproteins through its secretory pathway.Trypanosoma brucei is a protozoan parasite with two main proliferative stages in its life cycle: the procyclic form that grows in the tsetse fly midgut, and the bloodstream form that causes African sleeping sickness in humans and nagana in cattle. The bloodstream form is covered in a densely packed layer of 5 × 10⁶ glycosylphosphatidylinositol (GPI)-anchored variant surface glycoprotein (VSG) dimers. This coat protects the parasites from the alternative pathway of complement-mediated lysis, shields other cell surface proteins from the host immune system, and by the process of antigenic variation allows these parasites to persist for long periods in the host bloodstream (16, 54). The trypanosome genome contains several hundreds of silent VSG genes, most of which are pseudogenes in subtelomeric arrays (40). T. brucei evades host-acquired immunity through differential activation of these genes, which encode immunologically distinct GPI-anchored glycoproteins with one to three N glycosylation sites (27, 43).Protein N glycosylation is the most common covalent protein modification in eukaryotic cells (25). N-glycans contribute to “quality control” in the endoplasmic reticulum (ER) through a series of oligosaccharide-processing and lectin-binding reactions that contribute to protein folding and the targeting of misfolded glycoproteins for degradation (24, 47, 58, 65). As nascent protein chains enter the ER lumen, they are modified covalently in most eukaryotes by the addition of the Glc₃Man₉GlcNAc₂ core glycan via the action of oligosaccharyltransferase (OST). After deglucosylation by α-glucosidases I (GI) and II (GII), misfolded glycoproteins can be reglucosylated in the ER by the UDP-Glc:glycoprotein glucosyltransferase (UGGT), recreating the same monoglucosylated trimming intermediate generated by GII (9, 64, 66). UGGT behaves as a sensor of glycoprotein conformation and is a key constituent of ER quality control (50, 61). Calnexin and calreticulin are ER-resident lectin-like quality control chaperones that recognize the monoglucosylated glycans on glycoproteins and help them to fold properly through their close association with the oxidoreductase ERp57 (49). On reaching the proper tertiary structure, the glycoproteins are still substrates of GII but no longer of UGGT. Properly folded molecules, thus liberated from the lectins, are then free to continue their transit to the Golgi apparatus (64). When exposure to the folding machinery in the ER is not sufficient to promote a native conformation, proteins are eventually degraded by ER-associated degradation (49, 64).Most eukaryotes under conditions of stress, such as heat shock, undergo an unfolded protein response (UPR) that is triggered by sensing unfolded proteins in the ER. The UPR typically leads to increased expression of ER quality control components, such as calnexin and calreticulin and the ER chaperone Gpr78/BiP, as well inhibition of protein synthesis and cell cycle arrest (53, 57, 60).In contrast to the situation in most other eukaryotes, none of the trypanosomatid dolichol-linked oligosaccharides are capped with glucose residues, as these parasites do not synthesize the sugar donor dolichol-phosphate-glucose for these reactions (41, 59). The mature dolichol-phosphate-oligosaccharide species used for transfer to protein vary according to trypanosomatid species (17, 51, 52, 56). Therefore, in these organisms, monoglucosylated glycans are exclusively formed through UGGT-dependent glucosylation (12). Furthermore, trypanosomatids lack calnexin, which binds and participates in the refolding of glucosylated proteins, and it has been suggested that differences in the N-glycan precursor have profound effects on N-glycan-dependent quality control of glycoprotein folding and ER-associated degradation (4). These protozoa do not present a conventional OST complex and express only the catalytic stt3 protein subunit that, at least for the Trypanosoma cruzi and Leishmania major enzymes, shows little specificity toward the structure of the dolichol-phosphate-oligosaccharide donor (4, 11, 26, 31, 32, 45). In the case of T. brucei, while the insect-dwelling procyclic form makes and transfers Man₉GlcNAc₂-phosphate-dolichol (1), previous work from our group showed that the bloodstream form of the parasite transfers both Man₉GlcNAc₂ and Man₅GlcNAc₂ to VSG in a site-specific manner (29). Regarding ER folding and quality control, although in vitro assays have shown that T. cruzi and higher eukaryotic UGGTs exclusively glucosylate high-mannose glycans in misfolded glycoproteins (66), in T. brucei the UGGT and GII enzymes use Man₅GlcNAc₂ and Glc₁Man₅GlcNAc₂, respectively, as their substrates in the processing of VSG variant 221 (VSG221) (29). However, it could not be concluded from that study whether this apparent preference for atypical biantennary Man₅GlcNAc₂ and Glc₁Man₅GlcNAc₂ structures reflected the substrate specificity of the enzymes or the location of the glycosylation site in the VSG polypeptide chain (30).In this work, we further analyze the specificity and function of the UGGT/GII quality control system of T. brucei by analyzing the non-VSG N-glycans of our α-GII null mutant and creating and characterizing a T. brucei UGGT null mutant.     

Neuropeptide regulators of the juvenile hormone biosynthesis (in vitro) in the beetle,Tenebrio molitor (Coleoptera,Tenebrionidae)

The genome of Tribolium castaneum encodes two allatostatin [AS type B; W(X)₆Wamide and AS type C; PISCF‐OH] and one allatotropin (AT) precursor, but no AS type A (FGLamide) (Tribolium Genome Sequencing Consortium, 2008: Nature 452:949–955). Here we studied the activity (in vitro) of peptides derived from these precursors on the synthesis/release of juvenile hormone (JH) III. The corpora cardiaca‐corpora allata (CC‐CA) complexes of adult females of another tenebrionid beetle, the mealworm Tenebrio molitor, were used. Incubating the gland complexes in a medium containing Trica‐AS B3 peptide, we showed that the peptide has allatostatic function in T. molitor. The activity of the type C AS depended on the age of the test animals and their intrinsic rate of JH III biosynthesis. The Trica‐AS C peptide inhibited the JH release from CA of 3‐day‐old females with a high intrinsic rate of JH synthesis, but activated JH release from the CA of 7‐day‐old females with a lower intrinsic rate of JH production. The allatotropin peptide (Trica‐AT) also activated the JH release from the CA of 7‐day‐old females in a dose‐dependent and reversible manner. Unexpectedly, a type A AS derived from the precursor of the American cockroach Periplaneta americana (Peram‐AS A2b) inhibited the JH release from the CA of younger and older females in the concentration range of 10^?8 to 10^?4 M, and the effects were fully reversible in the absence of peptide. These data suggest a complex role of allatoactive neuropeptides in the regulation of JH III biosynthesis in beetles. © 2010 Wiley Periodicals, Inc.     

A regio- and stereo-controlled approach to triazoloquinoxalinyl C-nucleosides

The synthesis of a new series of acyclic triazoloquinoxalinyl C-nucleosides and their transformation to their cyclic analogs are described following protection, activation, and deprotection with subsequent intramolecular nucleophilic substitution protocol. The antibacterial potency of the new compounds was determined using an inhibition zone diameter test. The results show that 3a and 2b exhibit good activity against Escherichiacoli and Candidaalbicans. On the other hand, the cyclic mesylated C-nucleoside 13 showed activity against the Gram-positive bacteria (Staphylococcusaureus) and antifungal activity against C. albicans.     

Use of bioluminescence imaging to monitor Campylobacter survival in chicken litter

Aim: The aim of this study was to develop a novel approach for characterizing the growth and persistence of Campylobacter in different poultry‐rearing environments. Specifically, we constructed bioluminescent Campylobacter strains and used them to monitor the survival of these pathogens in litter (bedding) material. Methods and Results: We inserted shuttle plasmids carrying the luminescence genes (luxCDABE) into C. jejuni and C. coli to construct bioluminescent strains of these pathogens. The strains were spiked into microcosms containing samples of litter‐washings and dry litter collected from different enclosures that housed broiler chickens. Our results show that C. jejuni and C. coli survived for at least 20 days in reused (old) litter while the growth of these pathogens was inhibited in clean (new) litter. Furthermore, our results suggest that the availability of nutrients and the condition of the litter (reused vs new) are important factors in the persistence of these pathogens. Conclusions: Reused litter can potentially predispose chickens to Campylobacter contamination and maintaining clean litter might reduce the incidences of colonization with these pathogens. Significance and Impact of the Study: Bioluminescence provided a simple, sensitive, and rapid approach for analysing the growth dynamics of Campylobacter. Using this technology, we highlighted the potential role of litter material in maintaining these pathogens in the chicken environment.     

Molecular dissection of the migrating posterior lateral line primordium during early development in zebrafish

Background  

Development of the posterior lateral line (PLL) system in zebrafish involves cell migration, proliferation and differentiation of mechanosensory cells. The PLL forms when cranial placodal cells delaminate and become a coherent, migratory primordium that traverses the length of the fish to form this sensory system. As it migrates, the primordium deposits groups of cells called neuromasts, the specialized organs that contain the mechanosensory hair cells. Therefore the primordium provides both a model for studying collective directional cell migration and the differentiation of sensory cells from multipotent progenitor cells.     

Derivation and characterisation of hESC lines from supernumerary embryos, experience from Odense, Denmark

The derivation and characterisation of human embryonic stem cells provides a source of pluripotent stem cells with potential for clinical applications. Utilising locally sourced embryos from two IVF clinics, we derived and characterised five new cell lines for use in a non-clinical setting. Analysis of clinical data showed that the majority of embryos (94.5%) failed to reach the blastocyst stage of development and of all embryos, regardless of developmental status, 248 embryos were needed to create one stem cell line. From the number of embryos (69) which developed to the blastocyst stage 8.7% developed into cell lines. Using outgrowth of the whole blastocyst, we derived five new, unreported cell lines in Odense, Denmark between 2005 and 2006. Characterisation was carried out using RT-PCR, staining, karyotyping, EB formation and teratoma formation. The KMEB hESC lines will, in the future, be made available through the UK Stem Cell Bank (http://www.ukstemcellbank.org.uk/).     

Minocycline Synergizes with N-Acetylcysteine and Improves Cognition and Memory Following Traumatic Brain Injury in Rats

Background

There are no drugs presently available to treat traumatic brain injury (TBI). A variety of single drugs have failed clinical trials suggesting a role for drug combinations. Drug combinations acting synergistically often provide the greatest combination of potency and safety. The drugs examined (minocycline (MINO), N-acetylcysteine (NAC), simvastatin, cyclosporine A, and progesterone) had FDA-approval for uses other than TBI and limited brain injury in experimental TBI models.

Methodology/Principal Findings

Drugs were dosed one hour after injury using the controlled cortical impact (CCI) TBI model in adult rats. One week later, drugs were tested for efficacy and drug combinations tested for synergy on a hierarchy of behavioral tests that included active place avoidance testing. As monotherapy, only MINO improved acquisition of the massed version of active place avoidance that required memory lasting less than two hours. MINO-treated animals, however, were impaired during the spaced version of the same avoidance task that required 24-hour memory retention. Co-administration of NAC with MINO synergistically improved spaced learning. Examination of brain histology 2 weeks after injury suggested that MINO plus NAC preserved white, but not grey matter, since lesion volume was unaffected, yet myelin loss was attenuated. When dosed 3 hours before injury, MINO plus NAC as single drugs had no effect on interleukin-1 formation; together they synergistically lowered interleukin-1 levels. This effect on interleukin-1 was not observed when the drugs were dosed one hour after injury.

Conclusions/Significance

These observations suggest a potentially valuable role for MINO plus NAC to treat TBI.     

Synthesis and activity of p-azidobenzoyloxyferricrocin, a photoactivatable analog of ferrichrome

p-azidobenzoyloxy desferriferricrocin (AF) 2, a photoactivatable analog of ferrichrome, was prepared by selective acylation of the serine group of ferricrocin 1 in two steps: transesterification of ferricrocin followed by demetallation. A model compound, (L) 2-benzyloxycarbonylamino-3-p-azidobenzoyloxy N-isopropyl propionamide 8, was separately synthesized in order to set up optimal transesterification conditions to avoid , -elimination or epimerization of serine. Binding of iron-loaded AF (FeAF) to the FhuA outer membrane receptor protein of Escherichia coli AB2847 was demonstrated by inhibition of ferrichrome transport, interference with the infection by the bacteriophage 80 and with killing of cells by albomycin and colicin M. FeAF transported iron only weakly which indicates that the photoaffinity moiety is incompatible with transport or intracellular iron release from the siderophore.     

Occurrence of Virulence and Antimicrobial Resistance Genes in Escherichia coli Isolates from Different Aquatic Ecosystems within the St. Clair River and Detroit River Areas

Although the number of Escherichia coli bacteria in surface waters can differ greatly between locations, relatively little is known about the distribution of E. coli pathotypes in surface waters used as sources for drinking or recreation. DNA microarray technology is a suitable tool for this type of study due to its ability to detect high numbers of virulence and antimicrobial resistance genes simultaneously. Pathotype, phylogenetic group, and antimicrobial resistance gene profiles were determined for 308 E. coli isolates from surface water samples collected from diverse aquatic ecosystems at six different sites in the St. Clair River and Detroit River areas. A higher frequency (48%) of E. coli isolates possessing virulence and antimicrobial resistance genes was observed in an urban site located downstream of wastewater effluent outfalls than in the other examined sites (average of 24%). Most E. coli pathotypes were extraintestinal pathogenic E. coli (ExPEC) pathotypes and belonged to phylogenetic groups B2 and D. The ExPEC pathotypes were found to occur across all aquatic ecosystems investigated, including riverine, estuarine, and offshore lake locations. The results of this environmental study using DNA microarrays highlight the widespread distribution of E. coli pathotypes in aquatic ecosystems and the potential public health threat of E. coli pathotypes originating from municipal wastewater sources.     

Control of proliferation by Bcl-2 family members

The anti-proliferative effect of Bcl-2 acts mainly at the level of the G0/G1 phase of the cell cycle. Deletions and point mutations in the bcl-2 gene show that the anti-proliferative activity of Bcl-2, can in some cases, be dissociated from its anti-apoptotic function. This indicates that the effect of Bcl-2 on cell cycle progression can be a direct effect and not only a consequence of its anti-apoptotic activity. Bcl-2 appears to mediate its anti-proliferative effect by acting on both signal transduction pathways (NFAT, ERK) and on specific cell cycle regulators (p27, p130).