The glycosylation and characterization of the candidate Gc macrophage activating factor

The vitamin D binding protein, Gc globulin, has in recent years received some attention for its role as precursor for the extremely potent macrophage activating factor (GcMAF). An O-linked trisaccharide has been allocated to the threonine residue at position 420 in two of the three most common isoforms of Gc globulin (Gc1s and Gc1f). A substitution for a lysine residue at position 420 in Gc2 prevents this isoform from being glycosylated at that position. It has been suggested that Gc globulin subjected sequentially to sialidase and galactosidase treatment generates GcMAF in the form of Gc globulin with only a single GalNAc attached to T420. In this study we confirm the location of a linear trisaccharide on T420. Furthermore, we provide the first structural evidence of the generation of the proposed GcMAF by use of glycosidase treatment and mass spectrometry. Additionally the generated GcMAF candidate was tested for its effect on cytokine release from macrophages in human whole blood.     

An Observational Study of Slimming Behavior in Denmark in 1992 and 1998

Objective: To elucidate how frequent weight‐loss attempts are made, the methods used to achieve weight loss, and the extent to which the outcome is positive. Research Methods and Procedures: Two independent interviews were conducted in 1992 and in 1998, each with 1200 randomly selected adult subjects. Each survey was designed to ensure an equal distribution of age, gender, and geographical regions in Denmark. Results: The proportion of subjects having attempted weight loss did not change from 1992 to 1998, although the prevalence of overweight and obesity increased from 1992 (overweight, 30%; obesity, 6%) to 1998 (overweight, 35%; obesity, 8%). Almost twice as many women (61%) than men (32%) had attempted weight loss (p < 0.0001). Slimming occurred more often in subjects <50 years (51%) than >50 years (39%) (p < 0.0001), although overweight and obesity were more frequent in the elderly. Over‐the‐counter diet pills or meal replacements were associated with a negative outcome of slimming treatment (p < 0.0001). Discussion: Approximately half of all adult Danes have attempted weight loss, particularly women and individuals <50 years. This finding is inconsistent with the fact that overweight and obesity are more prevalent in men and in individuals >50 years. Changes in habitual diet and increased physical activity are the most prevalent modes of slimming, whereas the use of over‐the‐counter diet pills or meal replacements has decreased from 1992 to 1998. This development may have a positive impact on future body‐ weight‐management strategies.     

Chemically modified oligonucleotides with efficient RNase H response

Ten different chemically modified nucleosides were incorporated into short DNA strands (chimeric oligonucleotides ON3-ON12 and ON15-ON24) and then tested for their capacity to mediate RNAse H cleavage of the complementary RNA strand. The modifications were placed at two central positions directly in the RNase H cleaving region. The RNA strand of duplexes with ON3, ON5 and ON12 were cleaved more efficiently than the RNA strand of the DNA:RNA control duplex. There seems to be no correlation between the thermal stability between the duplexes and RNase H cleavage.     

Selenoprotein T is a novel OST subunit that regulates UPR signaling and hormone secretion

Selenoprotein T (SelT) is a recently characterized thioredoxin‐like protein whose expression is very high during development, but is confined to endocrine tissues in adulthood where its function is unknown. We report here that SelT is required for adaptation to the stressful conditions of high hormone level production in endocrine cells. Using immunofluorescence and TEM immunogold approaches, we find that SelT is expressed at the endoplasmic reticulum membrane in all hormone‐producing pituitary cell types. SelT knockdown in corticotrope cells promotes unfolded protein response (UPR) and ER stress and lowers endoplasmic reticulum‐associated protein degradation (ERAD) and hormone production. Using a screen in yeast for SelT‐membrane protein interactions, we sort keratinocyte‐associated protein 2 (KCP2), a subunit of the protein complex oligosaccharyltransferase (OST). In fact, SelT interacts not only with KCP2 but also with other subunits of the A‐type OST complex which are depleted after SelT knockdown leading to POMC N‐glycosylation defects. This study identifies SelT as a novel subunit of the A‐type OST complex, indispensable for its integrity and for ER homeostasis, and exerting a pivotal adaptive function that allows endocrine cells to properly achieve the maturation and secretion of hormones.     

Identification of protein-protein interfaces implicated in CD80-CD28 costimulatory signaling

The B7 ligands CD80 and CD86 on APCs deliver either costimulatory or inhibitory signals to the T cell when interacting with their counter-receptors CD28 and CD152 (CTLA-4) on the T cell surface. Although crucial for lymphocyte regulation, the structural basis of these interactions is still not completely understood. Using multivalent presentation and conditions mimicking clustering, believed to be essential for signaling through these receptors, and by applying a combined differential mass spectrometry and structural mapping approach to these conditions, we were able to identify a putative contact area involving hydrophilic regions on both CD28 and CD80 as well as a putative CD28 oligomerization interface induced by B7 ligation. Analysis of the CD80-CD28 interaction site reveals a well-defined interface structurally distinct from that of CD80 and CD152 and thus provides valuable information for therapeutic intervention targeted at this pathway, suggesting a general approach for other receptors.     

Cinnamic amides of (S)-2-(aminomethyl)pyrrolidines are potent H3 antagonists

New imidazole-free H3 antagonists have been found in a series of cinnamic amides of (S)-(aminomethyl)pyrrolidines. The influence of the substituent on the aromatic moiety on the potency and the inhibition of three cytochrome P450 subtypes are also described.     

Numerical chromosome errors in day 7 somatic nuclear transfer bovine blastocysts

Day 7 bovine somatic nuclear transfer (NT) embryos reconstructed from granulosa cells were examined for numerical chromosome aberrations as a potential cause of the high embryonic and fetal loss observed in such embryos after transfer. The NT embryos were reconstructed using a zona-free manipulation method: half-cytoplasts were made from zona-free oocytes by bisection, after which two half-oocytes and one granulosa cell (serum-starved primary culture) were fused together and activated. The NT embryos were cultured in modified synthetic oviductal fluid containing essential and nonessential amino acids, myoinositol, sodium citrate, and 5% cattle serum in microwells for 7 days, at which time nuclei from all blastocysts were extracted and chromosome aberrations were evaluated using dual-color fluorescent in situ hybridization with bovine chromosome 6- and 7-specific probes. Five embryo clone families, consisting of 112 blastocysts reconstructed from five different primary granulosa cell cultures, were examined. Overall, the mean chromosome complement within embryos was 86.9 +/- 3.7% (mean +/- SEM) diploid, 2.6 +/- 0.5% triploid, 10.0 +/- 3.1% tetraploid, and 0.5 +/- 0.2% pentaploid or greater; the vast majority (>75%) of the abnormal nuclei were tetraploid. Completely diploid and mixoploid embryos represented 22.1 +/- 4.5% and 73.7 +/- 5.5%, respectively, of all clones. Six totally polyploid blastocysts, containing or=5N chromosome complements, respectively) between two clone families were different (P < 0.01), as were blastocyst yields between other clone families (P < 0.01). Blastocyst yield was not correlated to % total ploidy error between clone families, but an inverse relationship (P < 0.01) between blastocyst total cell number and total % chromosome abnormality was observed within embryos. Categorization of the blastocysts into three quality grades (good, medium, and poor) and comparison of the distribution of ploidies when classified into 0%, 0.1-5.0%, 5.1-10.0%, 10.1-15.0%, and 15.1-100% errors within embryos indicated that medium- and poor-grade embryos were different (P < 0.05) from good-quality, in vitro-produced embryos. In a separate study, 11 different granulosa cell cultures (that did not correspond to those used for NT) were evaluated and found to possess only 0.23 +/- 0.12% ploidy errors. These results demonstrate that 1) the percentage of ploidy errors in bovine NT blastocysts is inversely related to total blastocyst cell number, 2) the mixoploid condition is representative of the majority of embryos, 3) 100% polyploid NT blastocysts can exist, and 4) the ploidy errors seem not to be derived from the donor cells.     

Density-dependence and within-host competition in a semelparous parasite of leaf-cutting ants

Background  

Parasite heterogeneity and within-host competition are thought to be important factors influencing the dynamics of host-parasite relationships. Yet, while there have been many theoretical investigations of how these factors may act, empirical data is more limited. We investigated the effects of parasite density and heterogeneity on parasite virulence and fitness using four strains of the entomopathogenic fungus, Metarhizium anisopliae var. anisopliae, and its leaf-cutting ant host Acromyrmex echinatior as the model system.