Biomaterial surface chemistry dictates adherent monocyte/macrophage cytokine expression in vitro

An in vitro human monocyte culture system was used to determine whether adherent monocyte/macrophage cytokine production was influenced by material surface chemistry. A polyethylene terephthalate (PET) base surface was modified by photograft copolymerization to yield hydrophobic, hydrophilic, anionic and cationic surfaces. Freshly isolated human monocytes were cultured onto the surfaces for periods up to 10 days in the presence or absence of interleukin-4 (IL-4). Semi-quantitative RT-PCR analysis on days 3, 7 and 10 of cell culture revealed that interleukin-10 (IL-10) expression significantly increased in cells adherent to the hydrophilic and anionic surfaces but significantly decreased in the cationic surface adherent monocytes/macrophages. Conversely, interleukin-8 (IL-8) expression was significantly decreased in cells adherent to the hydrophilic and anionic surfaces. Further analysis revealed that the hydrophilic and anionic surfaces inhibited monocyte adhesion and IL-4-mediated macrophage fusion into foreign body giant cells (FBGCs). Therefore, hydrophilic and anionic surfaces promote an anti-inflammatory type of response by dictating selective cytokine production by biomaterial adherent monocytes and macrophages. These studies contribute information necessary to enhance our understanding of biocompatibility to be used to improve the in vivo lifetime of implanted medical devices and prostheses.     

Tetrameric Detergent-Soluble Acetylcholinesterase from Human Caudate Nucleus: Subunit Composition and Number of Active Sites

Purified tetrameric detergent-soluble acetylcholinesterase (DS-AChE) from human caudate nucleus was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis in the absence as well as in presence of a reducing agent. Staining for protein revealed a main band at 66,000 daltons (light monomer) with additional bands at 78,000 daltons (heavy monomer) as well as 130,000 and 150,000 daltons (light and heavy dimers). The same four polypeptides were also detected by Western blotting and by autoradiography of [3H]diisopropylphosphoryl enzyme. Labeling of the enzyme with 3-trifluoromethyl-3-(m-[125I]-iodophenyl)diazirine showed that the heavy monomer contained the hydrophobic anchor of the enzyme, whereas the light monomer was practically not labeled. The hydrophobic anchor was susceptible to proteolytic degradation by proteinase K. The functional molarity of DS-AChE was determined by two independent methods. Four active sites for the tetrameric enzyme were estimated. The turnover number per site was 1.7 X 10(7) mol of acetylthiocholine iodide hydrolyzed X h-1.     

Diurnal and temporal changes in the chemical profile of xylem exudate from Vitis rotundifolia

Positive root pressure in Vitis rotundifolia Michx. cv. Noble was employed to quantify diurnal and temporal changes in the chemical profile of xylem exudate. Xylem fluid osmolarity (7.2 to 16.8 m M ), water flux (8.2 to 18.5 ml h⁻¹) and solute flux (0.7 to 2.2 mmol h⁻¹) from a cut spur exhibited a diurnal pattern with maxima during midday and minima at night. Total osmolarity was similar to the sum of all organic and inorganic entitites quantified, indicating that the major solutes have been identified. Total amino acid and organic acid concentration were about equal (2 to 7 m M ), and sugars accounted for a minor fraction of the total profile (<0.2 m M ). Glutamine represented ca 80% of the organic N and 70% of the total N transported in the xylem fluid. A circadian rhythm in water flux and net flux of most organic and inorganic entities was observed with maxima during midday and minima at night. The increase in xylem fluid osmolarity occurring during midday was primarily a consequence of increased organic acid (oxalic, citric, tartaric, malic and succinic acids) and ion (NH₄⁺, No₃⁻, P and Ca) concentration. A diurnal cycle in amino acid concentration was less clear. The concentration of individual organic and inorganic entities varied asynchronously with time. Xylem solute was comprised of 80% organic and 20% inorganic components when collected 5 min to 2 h after the commencement of bleeding, but the ratio of organic to inorganic components fell to about 50% after 7 days.     

Metabolism of amino acids, organic acids and sugars extracted from the xylem fluid of four host plants by adult Homalodisca coagulata

The efficiency of amino acid, organic acid and sugar metabolism was quantified for adult Homalodisca coagulata (Say) (Homoptera: Cicadellidae) by comparing chemical profiles of xylem fluid (food source) and insect exudate. Leafhoppers were confined in Parafilm^® sachets to stems of 4 host plants: [Baccharis halimifolia (L.), Lagerstroemia indica (L.), Prunus salicina (Lindl.), Prunus persica (L.), Batsch]. Insect feeding rates (0.09–0.27 ml h^–1), exudate osmolarity (7.8–12.8 mM) and exudate composition (mainly inorganic entities) were characteristic of a xylem feeder. Total organic solute concentration in the xylem fluid of B. halimifolia, L. indica, P. salicina and P. persica was ca. 9.4, 13.8, 5.5 and 1.8 mM, respectively. Nineteen protein amino acids, 7 organic acids and 3 or 4 sugars were identifid in the xylem fluid. Total amino acids, organic acids and sugars were metabolized with ca. 99% efficiency. Glutamine, asparagine, arginine and citric, malic and succinic acids, the predominant organic compounds in the xylem fluid of all four plant species, were metabolized with greater than 99% efficiency. Cysteine (51%), methionine (74%) and oxalic acid (77%) were metabolized with the lowest efficiency. The primary nitrogenous waste was NH _inf4 ^sup+ ; uric acid or urea were not detected. Nitrogen retention was generally less than 60% of dietary nitrogen. High feeding rates, ammonotelism and an extremely high metabolic efficiency of organic compounds permit H. coagulata to subsist on the dilute and skewed chemical profile of xylem fluid.

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Résumé L'efficacité du métabolisme des amino-acides, des acides organiques et des sucres a été quantifiée chez des H. coagulata Say (Hom. Cicadellidae) adultes, en comparant la composition chimique de la sève du xylème et du miellat des insectes. Les cicadelles ont été maintenues dans des sachets de parafilm avec des tiges de 4 plantes hôtes: Baccharis halimifolia L., Lagerstroemia indica L., Prunus salicina Lindl. et Prunus persica Batsch. Le taux de consommation (0,09 à 0,27 ml hr^–1), l'osmolarité du miellat (7,8 à 12,8 mM) et la composition du miellat (principalement des éléments inorganiques) sont caractéristiques des consommateurs de xylème. Les concentrations organiques totales en soluté de la sève du xylème étaient respectivement: 9,4; 13,8; 5,5; et 1,8 mM chez B. halimifolia, L. indica, P. salicina et P. persica. 19 amino acides protéiques, 7 acides organiques et 3 ou 4 sucres ont été identifiés dans la sève du xylème. Les acides aminés, les acides organiques et les sucres ont été métabolisés dans leur ensemble avec une efficacité de 99%. La glutamine, l'asparagine, l'arginine et les acides citrique, malique et succinique,-les principaux composés organiques de la sève du xylème de ces 4 plantes,-ont été métabolisés avec plus de 99% d'efficacité. La cystéine (51%), la méthionine (74%) et l'acide oxalique (77%) ont été métabolisés avec une plus faible efficacité. Le déchet azoté primarie était NH _inf4 ^sup+ ; l'acide urique et lurée n'ont pas été décelés. La fixation d'azote a été généralement inférieure à 60% de l'azote consommé. Des taux de consommation élevés, l'ammonotélisme et une efficacité extrêmement élevée du métabolisme des composés organiques permettent à H. coagulata de survivre malgré la composition chimique biaisée et la dilution de la sève du xylème.

     

Cholinesterase solubilizing factor from Cytophaga sp. is a phosphatidylinositol-specific phospholipase C

In the culture supernatant of Cytophaga sp. we detected an enzyme that converted glycosylphosphatidyl-inositol-anchored acetylcholinesterase to the hydrophilic form. This enzyme had a cleavage specificity of a phospholipase C. It hydrolyzed phosphatidylinositol but did not act on phosphatidylcholine. On gel filtration the enzyme migrated with an apparent molecular mass of about 17 kDa. It displayed maximal activity between pH 6-6.5 and did not require cofactors for the expression of catalytic activity. Mercurials and zinc ions inhibited the enzyme and its activity also decreased with increasing ionic strength in the assay. With acetylcholinesterase as substrate optimal activity was obtained in pure micelles of Triton X-100, whereas in mixed micelles containing Triton X-100 and phosphatidylcholine the activity was reduced. The enzyme from Cytophaga sp. showed little activity towards acetylcholinesterase embedded in intact membranes where more than 1000-times higher concentrations of phosphatidylinositol-specific phospholipase C was necessary to solubilize acetylcholinesterase as compared to acetylcholinesterase in detergent micelles.     

Isolation and characterization of a phosphatidylinositol-glycan-anchor-specific phospholipase D from bovine brain

In recent years an increasing number of proteins has been shown to be membrane-anchored by a covalently attached PtdIns-glycan residue. In mammalian cells little is known about PtdIns-glycan-specific phospholipases which might play a role in the metabolism of PtdIns-glycan-anchored proteins. In order to identify PtdIns-glycan-specific phospholipases, a rapid and sensitive assay for such enzymes was developed using the PtdIns-glycan-anchored amphiphilic membrane form of acetylcholinesterase as substrate. The rate of product formation was monitored by the increase in soluble hydrophilic acetylcholinesterase in the aqueous phase after separation in Triton X-114. With this assay we established the presence of a PtdIns-glycan-specific phospholipase in bovine brain. This enzyme was soluble and could be partially purified by a heat step followed by chromatography on DEAE-cellulose and by gel filtration on Sepharose CL-6B. PtdIns-glycan-specific phospholipase had a high affinity for the PtdIns-glycan anchor of the substrate (Km = 52 nM) and did not degrade either PtdCho or PtdIns. Hydrophobic labeling of the anchor of the substrate with 3-trifluoromethyl-3-(m-[125I]iodophenyl)diazirine [( 125I]TID) caused a marked decrease in the cleavage rate and methylation of the amino group of the glucosamine residue of the anchor decreased the cleavage rate to zero. Using [125I]TID-labeled substrate, diradylglycerol phosphate was identified as the second product showing that the cleavage specificity of PtdIns-glycan-specific phospholipase was that of a phospholipase D. PtdIns-glycan-specific phospholipase D was inhibited by mercurials, omicron-phenanthroline and EGTA. It was stimulated by Ca2+ in micromolar concentrations indicating that PtdIns-glycan-phospholipase D is a Ca2(+)-regulated enzyme.     

No distinct barrier effects of highways and a wide river on the genetic structure of the Alpine newt (<Emphasis Type="Italic">Ichthyosaura alpestris</Emphasis>) in densely settled landscapes

Linear landscape elements such as roads, railways and rivers have been shown to act as barriers to dispersal and gene flow, hence impeding functional connectivity and increasing genetic differentiation between individuals or populations on opposite sides of the barrier. Such putative barriers act through a confluence of mechanisms, including crossing mortality, barrier avoidance and modifications to organisms’ effective dispersal patterns. Small, terrestrial animals such as amphibians are predicted to be vulnerable to the effects of such barriers given their limited locomotive performance and their dependence on spatially distinct breeding habitats. Here, we examined the effects of highways and a wide river on Ichthyosaura alpestris in three regions of northern Switzerland by measuring the genetic differentiation between local populations and describing the spatial genetic structure. Moreover, we estimated effective population sizes as an indicator for the susceptibility of populations to random genetic drift. Based on genetic differentiation, we found evidence to suggest that the highways and river acted as barriers to gene flow for the newt in the study regions, but results were inconsistent when ignoring breeding ponds with low samples sizes. Admixture-based genetic clustering suggested the delineation of the genotypes to rough regional clusters, with only weak structure inferred within these clusters. Thus, results suggest that at present, highways and rivers do not substantially affect the genetic structure of I. alpestris within northern Switzerland in a negative manner. Alternatively, the lack of a distinct genetic structure in regional newt populations may be explained by, e.g., large effective population sizes.     

Quantitative composition and characterization of the proteins in membrane vesicles released from erythrocytes by dimyristoylphosphatidylcholine. A membrane system without cytoskeleton

Membrane vesicles were prepared by incubation of human erythrocytes with dimyristoylphosphatidylcholine [3] and isolated by isopycnic centrifugation on Dextran density gradients. Protein analyses were carried out with crossed immunoelectrophoresis and dodecylsulfate polyacrylamide gel electrophoresis. The right-side-out-oriented membrane vesicles contained membrane and cytoplasmic proteins of the erythrocyte but lacked cytoskeletal components. Comparison of proteins in vesicles and erythrocyte membranes showed that acetylcholinesterase was enriched two to six times in the vesicles relative to both membrane-spanning proteins, band 3, and glycophorin. Two further, hitherto unidentified, sialic acid-containing membrane antigens were found in the vesicles. Both faced the outside of the membranes and were enriched two to seven times. Ankyrin was not present in the membrane vesicles and spectrin could not be detected by dodecylsulfate polyacrylamide gel electrophoresis. We suggest that the redistribution of proteins in the vesicles reflects differences in their interactions with other membrane components and their relative mobility within the erythrocyte membrane.     

Purification from bee venom of melittin devoid of phospholipase A2 contamination

Conditions for the inactivation of phospholipase A₂ which contaminates melittin preparations were studied. A method for the purification of that peptide from bee venom is proposed. It gives, with a high recovery, a product devoid of phospholipase A₂ activity. In the first step, the venom is fractionated by gel filtration. Then the phospholipase A₂ still present in the melittin fraction is destroyed by sequential sulfitolysis and cyanogen bromide cleavage. This leaves the melittin intact. The final cation-exchange chromatography yields an homogeneous melittin preparation as analyzed by gel filtration, reverse-phase HPLC, and amino acid analysis.