The glycan processing and site occupancy of recombinant Thy-1 is markedly affected by the presence of a glycosylphosphatidylinositol anchor

Thy-1 is a cell surface glycoprotein containing three N-linked glycosylation sites and a glycosylphosphatidylinositol (GPI) anchor. The effect of the anchor on its N-linked glyco-sylation was investigated by comparing the glycosylation of soluble recombinant Thy-1 (sThy-1) with that of recombinant GPI anchored Thy-1, both expressed in Chinese hamster ovary cells. The sThy-1 was produced in a variety of isoforms including some which lacked carbohydrate on all three sequons whereas the GPI anchored form appeared fully glycosylated like native Thy-1. This was surprising as the attachment of N-linked sugars occurs cotranslationally and it was not expected that the presence of a C-terminal GPI anchor signal sequence would affect sequon occupancy. Furthermore sThy-1 lacking glycosylation could be produced with the inhibitor tunicamycin but in contrast cell surface expression of unglycosylated GPI anchored Thy-1 could not be obtained. The GPI anchored form appeared less processed with almost 4-fold more oligo-mannose oligosaccharides than in sThy-1 and also with less sialylated and core fucosylated biantennary glycans. Possible mechanisms whereby the anchor or the anchor signal sequence, control site occupancy and maturation are discussed.     

Assembly and characterization of heterochromatin and euchromatin on human artificial chromosomes

Background

Human centromere regions are characterized by the presence of alpha-satellite DNA, replication late in S phase and a heterochromatic appearance. Recent models propose that the centromere is organized into conserved chromatin domains in which chromatin containing CenH3 (centromere-specific H3 variant) at the functional centromere (kinetochore) forms within regions of heterochromatin. To address these models, we assayed formation of heterochromatin and euchromatin on de novo human artificial chromosomes containing alpha-satellite DNA. We also examined the relationship between chromatin composition and replication timing of artificial chromosomes.

Results

Heterochromatin factors (histone H3 lysine 9 methylation and HP1α) were enriched on artificial chromosomes estimated to be larger than 3 Mb in size but depleted on those smaller than 3 Mb. All artificial chromosomes assembled markers of euchromatin (histone H3 lysine 4 methylation), which may partly reflect marker-gene expression. Replication timing studies revealed that the replication timing of artificial chromosomes was heterogeneous. Heterochromatin-depleted artificial chromosomes replicated in early S phase whereas heterochromatin-enriched artificial chromosomes replicated in mid to late S phase.

Conclusions

Centromere regions on human artificial chromosomes and host chromosomes have similar amounts of CenH3 but exhibit highly varying degrees of heterochromatin, suggesting that only a small amount of heterochromatin may be required for centromere function. The formation of euchromatin on all artificial chromosomes demonstrates that they can provide a chromosome context suitable for gene expression. The earlier replication of the heterochromatin-depleted artificial chromosomes suggests that replication late in S phase is not a requirement for centromere function.

     

Signals and targets of the self-incompatibility response in pollen of Papaver rhoeas

Self-incompatibility (SI) in Papaver rhoeas involves an allele-specific recognition between stigmatic S-proteins and pollen, resulting in inhibition of incompatible pollen. A picture of some of the signalling events and mechanisms involved in this specific inhibition of pollen tube growth is beginning to be built up. This highly specific response triggers a Ca(2+)-dependent signalling cascade in incompatible pollen when a stigmatic S-protein interacts with it. Rapid increases in cytosolic free Ca(2+) concentration ([Ca(2+)](i)) can now be attributed (at least in part) to Ca(2+) influx. The rapid loss of the pollen apical Ca(2+) gradient within approximately 1-2 min is accompanied by the inhibition of pollen tube tip growth. Concomitant with this time-frame, hyper-phosphorylation of p26, a soluble pollen phosphoprotein is detected. Characterization of p26 reveals that it is a soluble inorganic pyrophosphatase, which suggests a possible direct functional role in pollen tube growth. Slightly later, a putative MAP kinase (p52) is thought to be activated. Finally, preliminary evidence that programmed cell death (PCD) may be triggered in this response is described. A key target for these signals, the actin cytoskeleton, has also been identified. In this article the current understanding of some of the components of this signalling cascade and how they are beginning to throw some light on possible mechanisms involved in this SI-induced inhibition of pollen tube growth, is discussed.     

O-glycan sialylation and the structure of the stalk-like region of the T cell co-receptor CD8

Studies of mucins suggest that the structural effects of O-glycans are restricted to steric interactions between peptide-linked GalNAc residues and adjacent polypeptide residues. It has been proposed, however, that differential O-glycan sialylation alters the structure of the stalk-like region of the T cell co-receptor, CD8, and that this, in turn, modulates ligand binding (Daniels, M. A., Devine, L., Miller, J. D., Moser, J. M., Lukacher, A. E., Altman, J. D., Kavathas, P., Hogquist, K. A., and Jameson, S. C. (2001) Immunity 15, 1051-1061; Moody, A. M., Chui, D., Reche, P. A., Priatel, J. J., Marth, J. D., and Reinherz, E. L. (2001) Cell 107, 501-512). We characterize the glycosylation of soluble, chimeric forms of the alphaalpha- and alphabeta-isoforms of murine CD8 containing the O-glycosylated stalk of rat CD8alphaalpha, and we show that the stalk O-glycans are differentially sialylated in CHO K1 versus Lec3.2.8.1 cells (82 versus approximately 6%, respectively). Sedimentation analysis indicates that the Perrin functions, Pexp, which reflect overall molecular shape, are very similar (1.61 versus 1.54), whereas the sedimentation coefficients (s) of the CHO K1- and Lec3.2.8.1-derived proteins differ considerably (3.73 versus 3.13 S). The hydrodynamic properties of molecular models also strongly imply that the sialylated and non-sialylated forms of the chimera have parallel, equally highly extended stalks ( approximately 2.6 A/residue). Our analysis indicates that, as in the case of mucins, the overall structure of O-glycosylated stalk-like peptides is sialylation-independent and that the functional effects of differential CD8 O-glycan sialylation need careful interpretation.     

Dopamine and alcohol relapse: D1 and D2 antagonists increase relapse rates in animal studies and in clinical trials

A considerable number of animal studies on the effects of dopaminergic agents on alcohol intake behavior have been performed. Acute alcohol administration in rats induces dopamine release in the caudate nucleus and in the nucleus accumbens, an effect related among others to reinforcement. It has been repeatedly suggested that D1 and D2 receptor activation mediates reward. As alcohol consumption and dopaminergic transmission seem to have a close relationship, all kinds of dopaminergic agents may be regarded as putative therapeutics for preventing relapse. In a prospective European double-blind multicenter clinical trial, comparing the D1, D2, D3 antagonist flupenthixol and placebo in 281 chronic alcohol-dependent patients (27.4% women), the application of the Lesch typology made an outcome differentiation possible. It could be shown in which patients flupenthixol administration was followed by a significantly higher relapse rate and in which patient groups no differences were found when compared to placebo.     

Intragastric ethanol infusion model for cellular and molecular studies of alcoholic liver disease

The intragastric alcohol infusion rat model (IAIRM) of alcoholic liver disease (ALD) has been utilized in various laboratories to study various aspects of ALD pathogenesis including oxidative stress, cytokine upregulation, hypoxic damage, apoptosis, ubiquitin-proteasome pathway and CYP2E1 induction. The basic value of the model is that it produces pathologic changes which resemble ALD including microvesicular and macrovesicular fat, megamitochondria, apoptosis, central lobular and pericellular fibrosis, portal fibrosis, bridging fibrosis, central necrosis, and mixed inflammatory infiltrate including PMNs and lymphocytes. The model is valuable because the diet and ethanol intake are totally under the control of the investigator. A steady state can be maintained with high or low blood alcohol levels for long periods. The cycling of the blood alcohol levels, when a constant infusion rate of alcohol is maintained, simulates binge drinking. Using this model the importance of dietary fat, especially the degree of saturation of the fatty acids on the induction of liver pathology, has been documented. The role of endotoxin, the Kupffer cell, TNFalpha, and NADPH oxidase have been demonstrated. The importance of 2E1 in oxidative stress induction has been shown using inhibitors of the isozyme. The importance of dietary iron in the pathogenesis of cirrhosis has been documented. Acetaldehyde has been shown to play a role in preventing liver pathology by preventing NFkappaB activation. Using the model, to maintain high blood alcohol levels is found to be necessary to demonstrate proteasomal peptidase inhibition. Ubiquitin synthesis is also inhibited at high blood alcohol levels in the IAIRM model. Oxidized proteins accumulate in the liver at high blood alcohol levels. Neoantigens derived from protein adducts formed with products of oxidation induce autoimmune mechanisms of liver injury. Thus, in many ways the model has revolutionized our understanding of the pathogenesis of ALD.     

Oxygen and nitrate-dependent regulation of <Emphasis Type="Italic">dmsABC</Emphasis> operon expression in <Emphasis Type="Italic">Escherichia coli</Emphasis>: sites for Fnr and NarL protein interactions

Background  

Escherichia coli can respire anaerobically using dimethyl sulfoxide (DMSO) or trimethylamine-N-oxide (TMAO) as the terminal electron acceptor for anaerobic energy generation. Expression of the dmsABC genes that encode the membrane-associated DMSO/TMAO reductase is positively regulated during anaerobic conditions by the Fnr protein and negatively regulated by the NarL protein when nitrate is present.     

Snipping polymorphisms from large EST collections in barley (<Emphasis Type="Italic">Hordeum vulgare </Emphasis>L.)

The public EST (expressed sequence tag) databases represent an enormous but heterogeneous repository of sequences, including many from a broad selection of plant species and a wide range of distinct varieties. The significant redundancy within large EST collections makes them an attractive resource for rapid pre-selection of candidate sequence polymorphisms. Here we present a strategy that allows rapid identification of candidate SNPs in barley (Hordeum vulgare L.) using publicly available EST databases. Analysis of 271,630 EST sequences from different cDNA libraries, representing 23 different barley varieties, resulted in the generation of 56,302 tentative consensus sequences. In all, 8171 of these unigene sequences are members of clusters with six or more ESTs. By applying a novel SNP detection algorithm (SNiPpER) to these sequences, we identified 3069 candidate inter-varietal SNPs. In order to verify these candidate SNPs, we selected a small subset of 63 present in 36 ESTs. Of the 63 SNPs selected, we were able to validate 54 (86%) using a direct sequencing approach. For further verification, 28 ESTs were mapped to distinct loci within the barley genome. The polymorphism information content (PIC) and nucleotide diversity () values of the SNPs identified by the SNiPpER algorithm are significantly higher than those that were obtained by random sequencing. This demonstrates the efficiency of our strategy for SNP identification and the cost-efficient development of EST-based SNP-markers.The first two authors contributed equally to this work