Structural and physicochemical requirements of endotoxins for the activation of arachidonic acid metabolism in mouse peritoneal macrophages in vitro

Lipopolysaccharides of different wild-type and mutant gram-negative bacteria, as well as synthetic and bacterial free lipid A, were studied for their ability to activate arachidonic acid metabolism in mouse peritoneal macrophages in vitro. It was found that lipopolysaccharides of deep-rough mutants of Salmonella minnesota and Escherichia coli (Re to Rc chemotypes) stimulated macrophages to release significant amounts of leukotriene C4 (LTC4) and prostaglandin E2 (PGE2). Lipopolysaccharides of wild-type strains (S. abortus equi, S. friedenau) only induced PGE2 and not LTC4 formation. Unexpectedly, free bacterial and synthetic E. coli lipid A were only weak inducers of LTC4 and PGE2 production. Deacylated Re-mutant lipopolysaccharide preparations were inactive. However, co-incubation of macrophages with both deacylated lipopolysaccharide and lipid A lead to the release of significant amounts of LTC4 and PGE2, similar to those obtained with Re-mutant lipopolysaccharide. The significance of the lipid A portion of lipopolysaccharide for the induction of LTC4 was indicated by demonstrating that peritoneal macrophages of endotoxin-low-responder mice or of mice rendered tolerant to endotoxin did not respond with the release of arachidonic acid metabolites on stimulation with Re-mutant lipopolysaccharide and that polymyxin B prevented the Re-lipopolysaccharide-induced LTC4 and PGE2 release. Physical measurements showed that the phase-transition temperatures of both free lipid A and S-form lipopolysaccharide were above 37 degrees C while those of R-mutant lipopolysaccharides were significantly lower (30-35 degrees C). Thus, with the materials investigated, an inverse relationship between the phase-transition temperature and the capacity to elicit LTC4 production was revealed.     

Insulin receptor internalization defect in an insulin-resistant mouse melanoma cell line

Previous studies from this laboratory demonstrated that the PG19 mouse melanoma cell line does not exhibit a biological response to insulin, whereas melanoma x mouse embryo fibroblast hybrids do respond to insulin. To investigate the molecular basis of the insulin resistance of the PG19 melanoma cells, insulin receptors from the insulin-resistant melanoma cells and insulin-sensitive fibroblast x melanoma hybrid cells were analyzed by the technique of photoaffinity labeling using the photoprobe 125I-NAPA-DP-insulin. Photolabeled insulin receptors from the two cell types have identical molecular weights as determined by SDS gel electrophoresis under reducing and nonreducing conditions, indicating that the receptors on the two cell lines are structurally similar. Insulin receptor internalization studies revealed that the hybrid cells internalize receptors to a high degree at 37 degrees C, whereas the melanoma cells internalize receptors to a very low degree or not at all. The correlation between ability to internalize insulin receptors and sensitivity to insulin action in this system suggests that uptake of the insulin-receptor complex may be required for insulin action in these cells. Insulin receptors from the two cell lines autophosphorylate in a similar insulin-dependent manner both in vitro and in intact cells, indicating that insulin receptors on the melanoma and hybrid cells have functional tyrosine protein kinase activity. Therefore, the block in insulin action in the PG19 melanoma cells appears to reside at a step beyond insulin-stimulated receptor autophosphorylation.     

Die reusenzelle (cyrtocyte) des Dinophilus (archiannelida)

Dinophilus has a protonephridial system with end-cells. These end-cells are cyrtocytes with an inner canal, a tuft of flagella and a cell wall consisting of rods joined by membranes.

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Abkürzungen BS Blutsinus - CC Cytoplasma der Cyrtocyte - CG Zellgrenze - CK Cytoplasma der Kanalzelle - CV Vertiefung der inneren Zelloberfläche an der Geißelbasis - G Geißel - Coe Coelom - MB Membran des Blutsinus - MC Membran um die Cyrtocyte-Basalmembran - MS Membran zwischen den Reusenstäben - NC Kern der Cyrtocyte - NK Kern der Kanalzelle - Pf Cytoplasmapfeiler der Cyrtocyte - Rs Reusenstab - Spd Speicheldrüse Senkrechte Linie in jeder Abbildung = 1      

Studies on the total synthesis of an A7,B7-dicarbainsulin. III. Assembly of segments and generation of biological activity

As a further contribution to the synthesis of an insulin analogue with a stable A7-B7 interchain bond, the synthesis of A(8-21) by solution methods, and of B(9-25) as well as [7-(2,7-diaminosuberic acid)]B(1-8) by solid phase methods is described. In the latter compound, the amino group of the diaminosuberic acid residue was acylated with A(1-6), and the resulting "U-peptide" sequentially elongated with the C-terminal A- and finally B-chain sequences. The conversion of the product into the disulfide moiety gave a mixture which could not be resolved by currently available methods. However, the low biological activity of the crude product indicates that the A7-B7 disulfide bond is not crucially important for the activity of insulin.     

Phase diagram of lipid A from Salmonella minnesota and Escherichia coli rough mutant lipopolysaccharide

We have reported here on the structural polymorphism of lipid A, the "endotoxic principle" of bacterial lipopolysaccharide. For lipid A of rough mutant lipopolysaccharide from Salmonella minnesota and Escherichia coli, the three-dimensional supramolecular structures were determined with x-ray diffraction utilizing synchrotron radiation. The investigations were performed in the water concentration range 10 to 95% by weight, at [lipid A]:[Mg2+] molar ratios from 1:0 to 0.1:1, and in the temperature range from 20 to 70 degrees C. These data were correlated with measurements of the beta----alpha phase behaviour which was monitored with differential scanning calorimetry and Fourier-transform infrared spectroscopy. We found that the transition temperature of the acyl chains ranges--in the absence of Mg2(+)-from 45 degrees C at high to 56 degrees C at low water content, and-at an equimolar content of Mg2(+)-from 52 degrees C at high to 59 degrees C at low water concentrations. In the gel phase-in which the lipid A acyl chains are more disordered than those from saturated phospholipids-cubic phases are adopted at high water content (greater than 60%) and at high [lipid A]:[Mg2+] molar ratios. At low water contents, lamellar states are assumed exclusively. In the liquid crystalline state of lipid A, the hexagonal HII state is adopted under all conditions. The structural variability of lipid A is highest at high water concentrations, and structural changes may be induced by only slight changes in temperature, water content, and Mg2+ concentration. Under physiological conditions, however, the lipid A assemblies exhibit a strong preference to cubic structures.     

Planar asymmetric lipid bilayers of glycosphingolipid or lipopolysaccharide on one side and phospholipids on the other: membrane potential, porin function, and complement activation.

We have determined some physicochemical properties of the monosaccharide-type fraction (GSL-1) of glycosphingolipids, the major glycolipid components of the outer leaflet of the Gram-negative species Sphingomonas paucimobilis. These properties included the state of order of the hydrocarbon moiety, the effective molecular area, surface charge density, and intrinsic transmembrane potential profile of reconstituted planar asymmetric GSL-1/phospholipid bilayer membranes. We have, furthermore, investigated the insertion into and the function of porin channels in the reconstituted bilayers and the complement-activating capability of GSL-1 surfaces. All results were compared with respective data for deep rough mutant lipopolysaccharide of Salmonella minnesota R595. We found a remarkable agreement in most functional properties of the two glycolipids.     

Insulin analogues with permuted A chain N-terminus (author's transl)]

By partial synthesis insulin analogues were prepared in which the amino acid in position 1 of the A chain was permuted. Glycine in position A 1 was exchanged for leucine, tert.- butyloxycarbonylvaline, valine, proline, lysine as well as glutamic acid. Two pathways of partial synthesis were followed: Firstly, des-1-glycine-A-chain S-sulfonate was reacted with active esters of tert.-butyloxycarbonylamino acids. The ensuing modified A-chains were combined with natural B-chain to give A1-permuted insulins. In the second procedure, the preparation of tris-Boc-[A1-leucine]insulin was accomplished by reaction of Boc-leucine N-hydroxysuccinimide ester with NalphaB1,NepsilonB29-bis(tert.-butyloxycarbonyl)-des-A1-glycine-insulin. The protected insulin derivative had been prepared by combination of des-glycine-A-chain with Nalpha1,Nepsilon29-bis(tert.-butyloxycarbonyl)-B-chain. The deprotected analogues differed considerably in their CD-spectra from insulin and possessed low in vitro biological activities of 2.5-17%. Crystallization attempts failed. Thus, the introduction of side chains in position A1 distorts the conformation sterically and decreases the biological activity.     

Risk,parental autonomy and the epistemic divide: preimplantation genetic diagnosis in the Australian print news media, 1990–2007

Preimplantation genetic diagnosis (PGD) is a reproductive/genetic technology which has become the subject of public and scholarly debate because it involves the evaluation and consequent selection (and implantation) or destruction of human embryos. This research investigates the way PGD is constituted in the Australian print news media. Foucauldian discourse analysis reveals that proponents draw on their direct knowledge and experience of PGD to support their claims. There is an epistemic divide between consumers and others claiming direct knowledge, and critics and others drawing on indirect or abstract understandings of PGD. This divide characterizes the discourses present in the data and is directly linked to changes in these over the period under analysis.     

Freshwater snail vital rates affected by non-lethal concentrations of silver nanoparticles

Freshwater gastropods are widespread and common members of benthic communities that interact with other species and conspecifics. Anthropogenic activities are increasing the presence of chemical contaminants in aquatic systems, which have the potential to disrupt species interactions through acute toxic effects and low-exposure chronic effects on vital rates and behaviors of organisms. We determined the effects of the commonly used manufactured nanomaterial, silver nanoparticles (AgNPs), on the survival, growth, reproduction, and behaviors of a common pulmonate gastropod, Physa acuta. Gastropod survival decreased in higher concentrations of AgNP (LC50 = 2.18 μg/l), but was enhanced when experimental containers included sediment (LC50 > 10 μg/l). Chronic exposures resulted in growth rates and size at first reproduction of snails declining in only the highest exposure concentration of 1 μg/l AgNP. Physa egg production was reduced by 50% when chronically exposed to 0.01 μg/l or greater AgNP. Physa crawled more rapidly when exposed to 0.01 μg/l AgNP and greater, indicating a stress response to higher AgNP concentrations. Physa exposed to 1 μg/l AgNP used near-surface habitats in a similar manner to those exposed to the threat of crayfish predation, indicating that the stress response to AgNP is similar in magnitude and direction to the threat of a predator. The sublethal effects reported here suggest that low but environmentally relevant concentrations of AgNP are likely to affect gastropod populations in many ways, potentially leading to measurable effects on communities and ecosystems.