The interaction of N-oleylethanolamine with phospholipid bilayers

Long chain acylamides of ethanolamine were previously found to increase in the infarcted canine myocardium. Subsequent in vitro experiments established a number of interesting biological and physiological properties of these compounds including alteration of rabbit skeletal sarcoplasmic reticulum function and inhibition of permeability dependent calcium release from heart mitochondria. These results suggested an interaction between the N-acylethanolamines and biological membranes. In the present work we show that the most potent species in previous studies, N-oleylethanolamine, forms stable complexes with phospholipid vesicles, lowers diphenylhexatriene polarization ratios in dimyristoylphosphatidylcholine and dipalmitoylphosphatidylcholine uni- and multilamellar bilayer vesicles, and also produces a concentration dependent decrease in the phase transitions of these lipid structures. In addition studies with parinaric acids also suggested that N-oleylethanolamine partitions preferentially into more fluid areas of the bilayer. The results are discussed in terms of possible effects on biological membranes.     

High-sensitivity amino acid analysis by derivatization with O-phthalaldehyde and 9-fluorenylmethyl chloroformate using fluorescence detection: applications in protein structure determination

An amino acid analysis method using a commercially available analyzer that accurately quantitates protein-derived amino acids in the 10-100 pmol range is described. The method utilizes the robotic capability of the analyzer's autosampler to perform precolumn derivatization of both primary and secondary amino acids with o-phthalaldehyde and 9-fluorenylmethyl chloroformate, respectively. The derivatized amino acids are then separated on a C-18 reverse-phase amino acid column and quantitated in a single run by fluorescence detection. The characterization of beta-lactoglobulin and two tryptic peptides from the bacterial enzyme diaminopimelic acid epimerase is used to demonstrate the sensitivity and utility of this method.     

SIMS microscopy in the biomedical field.

We attempted to indicate the requirements for biomedical applications of SIMS microscopy. Sample preparation methodology should preserve both the structural and the chemical integrity of the tissue. Furthermore, it is often necessary to correlate ionic and light microscope images. This implies a common methodological approach to sample preparation for both microscopes. The use of low or high mass resolution depends on the elements studied and their concentrations. To improve the acquisition and processing of images, digital imaging systems have to be designed and require both ionic and optical image superimposition. However, the images do not accurately reflect element concentration; a relative quantitative approach is possible by measuring secondary ion beam intensity. Using an internal reference element (carbon) and standard curves the results are expressed in micrograms/mg of tissue. Despite their limited lateral resolution (0.5 microns) the actual SIMS microscopes are very suitable for the resolution of biomedical problems posed by action modes and drug localization in human pathology. SIMS microscopy should provide a new tool for metabolic radiotherapy by facilitating dose evaluation. The advent of high lateral resolution SIMS imaging (less than 0.1 microns) should open up new fields in biomedical investigation.     

Peptide-N 4-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity could explain the occurrence of extracellular xylomannosides in a plant cell suspension

We have previously isolated mannoside and xylomannoside oligosaccharides with one or two terminal reducingN-acetylglucosamine residues from the extracellular medium of white campion (Silene alba) suspension culture. We have now demonstrated the presence of peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase (PNGase) activity in cell extracts as well in the culture medium that could explain the production of those compounds. An additional xylomannoside, (GlcNAc)Man₃(Xyl)GlcNAc(Fuc)GlcNAc, was characterized, and¹H- and¹³C-NMR assignments for the oligosaccharide Man₃(Xyl)GlcNAc(Fuc)GlcNAc were obtained using homonuclear and heteronuclear spectroscopy (COSY).Abbreviations Endo endo--N-acetylglucosaminidase - Fuc fucose - GlcNAc N-acetylglucosamine - Man mannose - NMR nuclear magnetic resonance - PNGase peptide-N ⁴-(N-acetylglucosaminyl)asparagine amidase - Xyl xylose     

Influence of maize root mucilage on soil aggregate stability

This study was undertaken to determine the effects of root exudates on soil aggregate stability. Root mucilage was collected from two-month old maize plants (Zea mays L.) Mucilage and glucose solutions were added at a rate of 2.45 g C kg⁻¹ dry soil to silty clay and silt loam soils. Amended soils, placed in serum flasks, were incubated for 42 d with a drying-wetting cycle after 21 d. Evolved CO₂ was measured periodically as well as the water-stable aggregates and soluble sugar and polysaccharide content of the soil. In mucilage-amended soils CO₂ evolution started with a lag phase of 2–3 days, which was not observed in glucose-amended soils. There was then a sharp increase in evolved CO₂ up to day 7. During the second incubation period there were only small differences in evolved C between treatments. Incorporation of mucilage in both soils resulted in a spectacular and immediate increase in soil aggregate stability. Thereafter, the percent of water-stable aggregates quickly decreased parallel to microbial degradation. On completion of the incubation, aggregate stability in the silty clay soil was still significantly higher in the presence of mucilage than in the control. This work supports the assumption that freshly released mucilage is able to stick very rapidly to soil particles and may protect the newly formed aggregates against water destruction. On the silty clay, microbial activity contributes to a stabilization of these established organo-mineral bounds.     

Heparin binding to lipoprotein lipase and low density lipoproteins

Heparin was fractionated on an affinity column of bovine milk lipoprotein lipase (LpL) immobilized to Affi-Gel-15. The bound heparin, designated high-reactive heparin (HRH), enhanced LpL activity, presumably by stabilizing the enzyme against denaturation. The unbound heparin fraction had no observable effect on the initial rate of enzyme activity. However, at longer times of incubation there was inhibition of LpL activity. LpL-specific HRH also showed a high, Ca²⁺-dependent precipitating activity towards human plasma low density lipoproteins (LDL). Since LpL and LDL both bind to heparin-like molecules at the surface of the arterial wall, we suggest that their similar heparin-binding specificity may have physiological consequences as it relates to the development of atherosclerosis.

Heparin binding Lipoprotein lipase LDL Apolipoprotein Lipolysis     

Integrated cultivation of salmonids and seaweeds in open systems

Bacterial abundance and production in a vertical profile in Lake Kariba (17dgS), Zimbabwe, were affected by solar irradiance. At the surface, 1.87 × 10⁹ bacteria 1^–1 were found and abundance peaked at 10 m (2.5 × 10⁹ bacteria l^-1), then decreasing with depth. Bacterial reproduction at the surface(0.145 µg C1^–1 h^–1) was nearly four times less than the production at 10 m although bacterial numbers were only 26% less. Thus, bacterial production per cell was lower at the surface than deeper down, suggesting that bacterial production is inhibited at the surface.Bacterial production in GF/F filtered lake water in Whirl Pack bags showed an exponential decrease down to 3 m depth. The inhibition was well in accordance with light extinction in the UV region. Phosphatase activity was low in light exposed bags compared to dark, indicating photolysis of extracellular enzymes, or phototransformation of recalcitrant DOM, which substitutes enzyme activity. Hypolimnetic enzyme activity was less affected by solar light than epilimnetic.     

Cold acclimation and photoinhibition of photosynthesis in Scots pine

Cold acclimation of Scots pine did not affect the susceptibility of photosynthesis to photoinhibition. Cold acclimation did however cause a suppression of the rate of CO₂ uptake, and at given light and temperature conditions a larger fraction of the photosystem II reaction centres were closed in cold-acclimated than in nonacclimated pine. Therefore, when assayed at the level of photosystem II reaction centres, i.e. in relation to the degree of photosystem closure, cold acclimation caused a significant increase in resistance to photoinhibition; at given levels of photosystem II closure the resistance to photoinhibition was higher after cold acclimation. This was particularly evident in measurements at 20° C. The amounts and activities of the majority of analyzed active oxygen scavengers were higher after cold acclimation. We suggest that this increase in protective enzymes and compounds, particularly Superoxide dismutase, ascorbate peroxidase, glutathione reductase and ascorbate of the chloroplasts, enables Scots pine to avoid excessive photoinhibition of photosynthesis despite partial suppression of photosynthesis upon cold acclimation. An increased capacity for light-induced de-epoxidation of violaxanthin to zeaxanthin upon cold acclimation may also be of significance.Abbreviations APX ascorbate peroxidase - DHA dehydroascorbate - DHAR dehydroascorbate reductase - Fm maximal fluorescence when all reaction centres are closed - Fv/Fm maximum photochemical yield of PSII - GR glutathione reductase - GSH reduced glutathione - Je rate of photosynthetic electron transport - MDAR monodehydroascorbate reductase - q_N nonphotochemical quenching of fluorescence - q_P photochemical quenching of fluorescence - SOD superoxide dismutase This work was supported by the Swedish Natural Science Research Council and the National Natural Science Foundation of China.     

Expression of a Synthetic Gene Encoding the Anticoagulant-Antimetastatic Protein Ghilanten by the Methylotropic YeastPichia pastoris

Ghilantens are a family of cysteine-rich inhibitors of the clotting enzyme, factor Xa, that are produced in the salivary glands of the South American leechHaementeria ghilianii.In this study, a gene, designed from the amino acid sequence of a specific ghilanten isoform, was assembled from eight double-stranded oligonucleotide fragments. A yeast expression plasmid, pPIC9HG-2, was constructed by making an in-frame fusion of the ghilanten-coding sequences with the region encoding the pre-pro α-mating factor signal sequence for secretion. The expression of ghilanten in pPIC9HG-2 was under the control of the methanol-inducible, alcohol oxidase (AOX1) promoter.Pichia pastorisyeast strains KM 71 and SMD 1168 were transformed with linearized pPIC9HG-2 to target integration of the plasmid to the chromosomal 5′-AOX1locus via homologous recombination. Both strains yielded His⁺transformants that secreted a potent anticoagulant activity into the medium. Product yield was improved by using buffered media (pH 6.0) supplemented with either casamino acids or a mixture of yeast extract and peptone. The protease-deficient strain, SMD 1168, secreted about a twofold higher level ofr-ghilanten than KM 71. Significant clonal variation in the expression ofr-ghilanten was found among the His⁺transformants. A high producing clone was selected for production at the 2-liter shake flask and 10-liter bioreactor scales.r-Ghilanten was recovered from the fermentation broths in a single step by heparin Sepharose affinity chromatography. Protein sequence analysis of the amino terminus showed that the correct processing to yield mature ghilanten varied with the fermentation conditions.