Alginate and pectin composite films crosslinked with Ca ions: Effect of the plasticizer concentration

The manufacture of composite biofilms of alginate and LM-pectin crosslinked with calcium ions requires a two-step contact with Ca²⁺: initially a low-structured pre-film is formatted which is further crosslinked in a second contact with a more concentrated Ca²⁺ solution containing plasticizer. This research evaluated the influence of the plasticizer (glycerol) concentration (1–15% w/v) in this finishing reticulation step on final films characteristics. The results indicated that the extent of the simultaneous Ca²⁺ crosslinking and plasticization with glycerol was determined by the level of structural organization obtained in the pre-reticulation. Increasing the glycerol concentration of the crosslinking solution increased film solubility in water, moisture content, volumetric swelling and flexibility and decreased the resistance to tensile stress. Transparent alginate and pectin composite films with acceptable mechanical properties, low solubility and limited degree of swelling were obtained with 10% glycerol in the second contact solution.     

The relationships of cholesterol metabolism and plasma plant sterols with the severity of coronary artery disease

Changes in the balance of cholesterol absorption and synthesis and moderately elevated plasma plant sterols have been suggested to be atherogenic. Measuring cholestanol, lathosterol, campesterol, and sitosterol, we investigated the relationships of cholesterol metabolism and plasma plant sterols with the severity of coronary artery disease (CAD) in 2,440 participants of the Ludwigshafen Risk and Cardiovascular health (LURIC) study. The coronary status was determined by angiography, and the severity of CAD was assessed by the Friesinger Score (FS). An increase in the ratio of cholestanol to cholesterol was associated with high FS (P = 0.006). In contrast, a high ratio of lathosterol to cholesterol went in parallel with low FS (P < 0.001). Whereas the campesterol to cholesterol ratio significantly correlated with the FS (P = 0.026), the relationship of the sitosterol to cholesterol ratio with the FS did not reach statistical significance in the whole group. Increased campesterol, sitosterol, and cholestanol to lathosterol ratios were associated high FS (P < 0.001). To conclude, there is a modest association of high cholesterol absorption and low cholesterol synthesis with an increased severity of CAD. An atherogenic role of plasma plant sterols themselves, however, seems unlikely in subjects without sitosterolaemia.     

Structural Basis of Thermal Stability of the Tungsten Cofactor Synthesis Protein MoaB from Pyrococcus furiosus

Molybdenum and tungsten cofactors share a similar pterin-based scaffold, which hosts an ene-dithiolate function being essential for the coordination of either molybdenum or tungsten. The biosynthesis of both cofactors involves a multistep pathway, which ends with the activation of the metal binding pterin (MPT) by adenylylation before the respective metal is incorporated. In the hyperthermophilic organism Pyrococcus furiosus, the hexameric protein MoaB (PfuMoaB) has been shown to catalyse MPT-adenylylation. Here we determined the crystal structure of PfuMoaB at 2.5 Å resolution and identified key residues of α3-helix mediating hexamer formation. Given that PfuMoaB homologues from mesophilic organisms form trimers, we investigated the impact on PfuMoaB hexamerization on thermal stability and activity. Using structure-guided mutagenesis, we successfully disrupted the hexamer interface in PfuMoaB. The resulting PfuMoaB-H3 variant formed monomers, dimers and trimers as determined by size exclusion chromatography. Circular dichroism spectroscopy as well as chemical cross-linking coupled to mass spectrometry confirmed a wild-type-like fold of the protomers as well as inter-subunits contacts. The melting temperature of PfuMoaB-H3 was found to be reduced by more than 15°C as determined by differential scanning calorimetry, thus demonstrating hexamerization as key determinant for PfuMoaB thermal stability. Remarkably, while a loss of activity at temperatures higher than 50°C was observed in the PfuMoaB-H3 variant, at lower temperatures, we determined a significantly increased catalytic activity. The latter suggests a gain in conformational flexibility caused by the disruption of the hexamerization interface.     

Cholesteryl ester hydrolase activity is abolished in HSL macrophages but unchanged in macrophages lacking KIAA1363

Cholesteryl ester (CE) accumulation in macrophages represents a crucial event during foam cell formation, a hallmark of atherogenesis. Here we investigated the role of two previously described CE hydrolases, hormone-sensitive lipase (HSL) and KIAA1363, in macrophage CE hydrolysis. HSL and KIAA1363 exhibited marked differences in their abilities to hydrolyze CE, triacylglycerol (TG), diacylglycerol (DG), and 2-acetyl monoalkylglycerol ether (AcMAGE), a precursor for biosynthesis of platelet-activating factor (PAF). HSL efficiently cleaved all four substrates, whereas KIAA1363 hydrolyzed only AcMAGE. This contradicts previous studies suggesting that KIAA1363 is a neutral CE hydrolase. Macrophages of KIAA1363^−/− and wild-type mice exhibited identical neutral CE hydrolase activity, which was almost abolished in tissues and macrophages of HSL^−/− mice. Conversely, AcMAGE hydrolase activity was diminished in macrophages and some tissues of KIAA1363^−/− but unchanged in HSL^−/− mice. CE turnover was unaffected in macrophages lacking KIAA1363 and HSL, whereas cAMP-dependent cholesterol efflux was influenced by HSL but not by KIAA1363. Despite decreased CE hydrolase activities, HSL^−/− macrophages exhibited CE accumulation similar to wild-type (WT) macrophages. We conclude that additional enzymes must exist that cooperate with HSL to regulate CE levels in macrophages. KIAA1363 affects AcMAGE hydrolase activity but is of minor importance as a direct CE hydrolase in macrophages.     

Nutritive value of peas for nonruminant diets

Peas, the seeds of Pisum sativum, are produced usually in temperate regions but are accepted as a food source worldwide. Traditionally, nonruminant diets utilized peas which had been rejected by the food industry but specific cultivars of feed (or field) peas also have been developed for livestock use. In view of the diversity of varieties, seeding times (spring or winter-sown) and agronomic conditions during the growing season, there is a considerable range in the composition and nutritive value of peas. The seed coat (hull) represents 70 to 140 g kg⁻¹ of the total weight and consists mainly of non-starch polysaccharides, while the major components of the dehulled pea are starch ( 450 g kg⁻¹) and protein ( 250 g kg⁻¹). Published energy values for the whole seed range from 12.2 to 16.6 MJ ME kg⁻¹ DM for pigs and 10.1 to 12.8 MJ TME_N kg⁻¹ DM for poultry. Reported analyses for crude protein vary from 156 to 325 g kg⁻¹ DM, while content and availability of the constituent amino acids vary also with cultivar, seed type and analytical methods. Most concerns about low digestibilities relate to the sulphur amino acids and tryptophan. Potentially detrimental constituents in raw peas include anti-proteases, haemagglutinins, phytic acid and tannins although these appear negligible in Canadian peas. When analytical data are lacking, the following limits to use of peas are suggested: 100, 200 and 350 g kg⁻¹, respectively, in pig starter, grower and finisher diets; 200 g kg⁻¹ in broiler, 250 g kg⁻¹ in turkey and 300 g kg⁻¹ in layer diets.     

Protein-Mediated Transformation of Lipid Vesicles into Tubular Networks

Key cellular processes are frequently accompanied by protein-facilitated shape changes in the plasma membrane. N-BAR-domain protein modules generate curvature by means of complex interactions with the membrane surface. The way they assemble and the mechanism by which they operate are largely dependent on their binding density. Although the mechanism at lower densities has recently begun to emerge, how membrane scaffolds form at high densities remains unclear. By combining electron microscopy and multiscale simulations, we show that N-BAR proteins at high densities can transform a lipid vesicle into a 3D tubular network. We show that this process is a consequence of excess adhesive energy combined with the local stiffening of the membrane, which occurs in a narrow range of mechanical properties of both the membrane and the protein. We show that lipid diffusion is significantly reduced by protein binding at this density regime and even more in areas of high Gaussian curvature, indicating a potential effect on molecular transport in cells. Finally, we reveal that the breaking of the bilayer topology is accompanied by the nematic arrangement of the protein on the surface, a structural motif that likely drives the formation of reticular structures in living cells.     

Control of formation of active soluble or inactive insoluble baker''s yeast α-glucosidase PI in Escherichia coli by induction and growth conditions

Summary Using standard growth conditions (LB medium, 37°C, induction with 5 mM IPTG) yeast -glucosidase PI expressed under the control of the regulated tac-hybrid promoter results in the synthesis of insoluble aggregated -glucosidase granules in Escherichia coli. Under these conditions active soluble -glucosidase amounts to less than 1% of the heterologously produced protein. However, the amount of soluble active -glucosidase was dramatically increased when the strong tac-hybrid promoter was to a limited extent induced. This was achieved at concentrations of 0.01 mM IPTG or of 1% lactose or lower in a lactosepermease deficient host strain containing the lacI ^qrepressor gene on an R-plasmid. The formation of active soluble -glucosidase was almost 100% when E. coli cells induced in this manner were cultivated under conditions that reduced growth rate, i.e. at decreased temperature, extreme pH values or in minimal and complete media supplemented with different carbon sources.Abbreviations IPTG isopropyl--D-thiogalactopyranoside - RB refractile body - t-PA tissue type plasminogen activator - IFN interferon - X--glucoside 5-bromo-4-chloro-indolyl--D-glucopyranoside     

A quantitative analysis of isotope concentration profiles and rapid transport velocities in the C-fibers of the garfish olfactory nerve

In the olfactory nerve of the long-nosed garfish (Lepisosteus osseus), unusually well-defined isotope concentration distributions can be established with the rapid transport process. Transport velocities of two profile loci can be accurately described and a quantitative profile analysis is possible after profile normalization. Results from such studies indicate that: (1) peak amplitudes decrease exponentially as a function of distance from the olfactory mucosa according to the equation p = 2130 exp (– 0.109x); (2) the wavefront base and the peak apex loci move at rates of 221 ± 2 and 201 ± 4 mm/day, respectively (at 23°C), revealing a peak dispersion or broadening during transport; (3) the broadening is asymmetric with material shifting to the rear of the peak; (4) plateau regions are established behind the peak with material deposited by the peak; (5) only 20% of the total radioactivity in a cut nerve reaches the nerve terminals in the rapid transport peak while 80% is deposited along the axon; (6) profile areas from cut nerves decrease and lose 15% of their activity in 20 hr, while intact nerve profiles increase 10% in 16 hr due to continued somal contribution to the profile; (7) the displacement of the wavefront base (WFB) and peak apex (PA) profile loci can be described by the functions (8) transport velocities are linear functions of temperature between 10 and 25°C and increase 370% in that range. A linear extrapolation of the WFB and PA functions to 37°C yields 410 and 377 mm/day, respectively.     

Chlamydomonas reinhardtii CNX1E reconstitutes molybdenum cofactor biosynthesis in Escherichia coli mutants

We have isolated and characterized the Chlamydomonas reinhardtii genes for molybdenum cofactor biosynthesis, namely, CNX1G and CNX1E, and expressed them and their chimeric fusions in Chlamydomonas and Escherichia coli. In all cases, the wild-type phenotype was restored in individual mutants as well as in a CNX1G CNX1E double mutant. Therefore, CrCNX1E is the first eukaryotic protein able to complement an E. coli moeA mutant.