Sub-micellar phospholipid accelerates amyloid formation by apolipoprotein C-II

Lipid-free human apolipoprotein C-II (apoC-II) forms amyloid fibrils with characteristic beta-structure. This conformation is distinct from the alpha-helical fold of lipid-bound apoC-II. We have investigated the effect of the short-chain phospholipid, dihexanoylphosphatidylcholine (DHPC) on amyloid formation by apoC-II. The alpha-helical content of apoC-II increases in the presence of micellar DHPC (16 mM) and amyloid formation is inhibited. However, at sub-micellar DHPC concentrations (below 8 mM) amyloid formation is accelerated 6 fold. These results suggest that individual phospholipid molecules in vivo may exert significant effects on amyloid folding pathways.     

Both CD4(+) T cells and CD8(+) T cells are required for iodine accelerated thyroiditis in NOD mice

The nonobese diabetic (NOD) mouse, a spontaneous animal model for insulin-dependent diabetes mellitus, displays a tendency in common with human diabetic populations to develop autoimmune thyroiditis although incidence and severity of thyroid lesions vary widely among different colonies around the world. A congenic strain of NOD mice bearing I-Ak on a NOD background (NOD-H2(h4)) has recently been derived and displays a much greater tendency to develop thyroiditis and autoantibodies to mouse thyroglobulin (MTg) although it is free of diabetes. Both thyroid infiltrates and autoantibody formation are accelerated and enhanced in NOD-H2(h4) mice by increased iodine intake. The effect of increased iodine intake on NOD mice themselves has not been directly investigated although a recent study of these animals given high or low doses of iodine showed no follicular destruction unless the mice were first rendered goitrous by iodine deprivation. We found that dietary iodine increased both the incidence and the severity of thyroid lesions in our NOD mice although autoantibodies to MTg were absent. NOD background genes appear to be essential for the development of these lesions, which were maximal after 4 weeks of iodine administration and showed no significant regression when the iodine was stopped. Furthermore, our studies show for the first time that both CD4(+) and CD8(+) T cells are necessary for the development of this accelerated but essentially spontaneous murine thyroid disease.     

Regulation of Gi by the CB1 cannabinoid receptor C-terminal juxtamembrane region: structural requirements determined by peptide analysis

A CB1 cannabinoid receptor peptide fragment from the C-terminal juxtamembrane region autonomously inhibits adenylyl cyclase activity in a neuroblastoma membrane preparation. The cannabinoid receptor antagonist, SR141716A, failed to block the response. The peptide was able to evoke the response in membranes from Chinese hamster ovary (CHO) cells that do not express the CB1 receptor. These studies are consistent with a direct activation of Gi by the peptide. To test the importance of a BXBXXB sequence, Lys403 was acetylated, resulting in a peptide having similar affinity but reduced efficacy. N-Terminal truncation of Arg401 resulted in a 6-fold loss of affinity, which was not further reduced by sequential truncation of up to the first seven amino acids, four of which are charged. N-Terminal-truncated peptides exhibited maximal activity, suggesting that Gi activation can be conferred by the remaining amino acids. Truncation of the C-terminal Glu417 or substitution of Glu417 by a Leu or of Arg401 by a Norleucine reduced activity at 100 microM. The C-terminal juxtamembrane peptide was constrained to a loop peptide by placement of Cys residues at both terminals and disulfide coupling. This modification reduced the affinity 3-fold but yielded near-maximal efficacy. Blocking the Cys termini resulted in a loss of efficacy. Circular dichroism spectropolarimetry revealed that all C-terminal juxtamembrane peptide analogues exist in a random coil conformation in an aqueous environment. A hydrophobic environment (trifluoroethanol) failed to induce alpha-helix formation in the C-terminal juxtamembrane peptide but did so in less active peptides. The anionic detergent sodium dodecyl sulfate induced alpha-helix formation in all analogues except the loop peptide, where it induces a left-handed PII conformation. It is concluded that alpha-helix formation is not required for Gi activation.     

Age-related hematologic changes in captive-reared houbara,white-bellied,and rufous-crested bustards

Blood samples were obtained at monthly intervals between April 1994 and March 1996 from captive-bred houbara (Chlamydotis undulata macqueenii), rufous-crested (Eupodotis ruficrista gindiana), and white-bellied (Eupodotis senegalensis) bustards from 4-24 wk of age. Hematology investigations were conducted to determine age-related changes and to establish reference values for growing chicks of these species. There were significant age-related changes in hematocrit, hemoglobin, and red cell count in young birds compared with those of adults. White cell counts (lymphocytes and monocytes) were higher in juvenile birds, compared with adult values.     

Acid Denaturation of alpha1-antitrypsin: characterization of a novel mechanism of serpin polymerization

The native serpin architecture is extremely sensitive to mutation and environmental factors. These factors induce the formation of a partially folded species that results in the production of inactive loop-sheet polymers. The deposition of these aggregates in tissue, results in diseases such as liver cirrhosis, thrombosis, angioedema and dementia. In this study, we characterize the kinetics and conformational changes of alpha(1)-antitrypsin polymerization at pH 4 using tryptophan fluorescence, circular dichroism, turbidity changes and thioflavin T binding. These biophysical techniques have demonstrated that polymerization begins with a reversible conformational change that results in partial loss of secondary structure and distortion at the top of beta-sheet A. This is followed by two bimolecular processes. First, protodimers are formed, which can be dissociated by changing the pH back to 8. Then, an irreversible conformational change occurs, resulting in the stabilization of the dimers with a concomitant increase in beta-sheet structure, allowing for subsequent polymer extension. Electron microscopy analysis of the polymers, coupled with the far-UV CD and thioflavin T properties of the pH 4 polymers suggest they do not form via the classical loop-beta-sheet A linkage. However, they more closely resemble those formed by the pathological variant M(malton). Taken together, these data describe a novel kinetic mechanism of serine proteinase inhibitor polymerization.     

Profound differences in leukocyte-endothelial cell responses to lipopolysaccharide versus lipoteichoic acid

We have investigated the effects of LPS from Escherichia coli, lipoteichoic acid (LTA), and peptidoglycan (PepG) from Staphylococcus aureus, and live S. aureus on leukocyte-endothelial interactions in vivo using intravital microscopy to visualize muscle microvasculature. Systemic vs local administration of LPS induced very different responses. Local administration of LPS into muscle induced significant leukocyte rolling, adhesion, and emigration in postcapillary venules at the site of injection. LPS given systemically dramatically dropped circulating leukocyte counts and increased neutrophils in the lung. However, the drop in circulating leukocytes was not associated with leukocyte sequestration to the site of injection (peritoneum) nor to peripheral microvessels in muscles. Unlike LPS, various preparations of LTA had no systemic and very minor local effect on leukocyte-endothelial interactions, even at high doses and for prolonged duration. LPS, but not LTA, potently activated human endothelium to recruit leukocytes under flow conditions in vitro. Endothelial adhesion molecule expression was also increased extensively with LPS, but not LTA. Interestingly, systemic administration of live S. aureus induced leukocyte-endothelial cell responses similar to LPS. PepG was able to induce leukocyte-endothelial interactions in muscle and peritoneum, but had no effect systemically (no increase in neutrophils in lungs and no decrease in circulating neutrophil counts). These results demonstrate that: 1) LPS has potent, but divergent local and systemic effects on leukocyte-endothelial interactions; 2) S. aureus can induce a systemic response similar to LPS, but this response is unlikely to be due to LTA, but more likely to be mediated in part by PepG.     

IL-11 and IL-11Rα immunolocalisation at primate implantation sites supports a role for IL-11 in placentation and fetal development

Embryo implantation, endometrial stromal cell decidualization and formation of a functional placenta are critical processes in the establishment and maintenance of pregnancy. Interleukin (IL)-11 signalling is essential for adequate decidualization in the mouse uterus and IL-11 promotes decidualization in the human. IL-11 action is mediated via binding to the specific IL-11 receptor α (IL-11Rα). The present study examined immunoreactive IL-11 and IL-11Rα in cycling rhesus monkey endometrium, at implantation sites in cynomolgus and rhesus monkeys and in human first trimester decidua and defined distinct spatial and temporal patterns. In cycling rhesus monkey endometrium, IL-11 and IL-11Rα increased in both basalis and functionalis regions during the secretory compared with the proliferative phase, with changing cellular locations in luminal and glandular epithelium and stroma. The patterns were similar overall to those previously described in human endometrium. Differences were seen in immunostaining during implantation in cynomologus and rhesus monkey. In the cynomolgus, very little staining for IL-11 or IL-11Rα was seen in syncytio- and cyto-trophoblast cells in the villi between days 12 and 150 of pregnancy although there was moderate staining in cytotrophoblast in the shell between days 12 and 17 and in subpopulations of cytotrophoblast cells invading the arteries at day 17. By contrast in the rhesus monkey between days 24 and 35 of pregnancy and in human first trimester placenta, cyto- and syncytio-trophoblast in the villi but not cytotrophoblast in the shell were positively stained. The most intense staining for both IL-11 and IL-11Rα was present within the decidua in the maternal component of implantation sites in all three primates but moderate staining was also present in maternal vascular smooth muscle and glands perivascular cells and epithelial plaques. These results are consistent with a role for IL-11 both during decidualization and placentation in primates.