Anaerobic degradation of papermill sludge in a two-phase digester containing rumen microorganisms and colonized polyurethane foam

Summary The use of reticulated polyurethane foam as a support material for the immobilization of methanogenic associations and its application to the anaerobic treatment of fine particulate solid wastes was investigated. The colonization of polyurethane support particles in a continuous upflow reactor fed on a mixture of acetate, propionate and butyrate, was both rapid and dense. The combination of rumen microorganisms and colonized support particles in a two-phase digester resulted in an efficient anaerobic decomposition of papermill sludge.     

Membrane-mediated assembly of the prothrombinase complex

Prothrombinase assembly was studied on macroscopic planar bilayers consisting of 20% dioleoyl-phosphatidylserine (DOPS) and 80% dioleoyl-phosphatidylcholine (DOPC). The dissociation constant for the binding of factor Xa to the bilayer, measured by ellipsometry, was Kd = 47 +/- 8 nM (mean +/- S.D.) and this value was lowered to Kd = 2.2 +/- 0.3 pM by preadsorption of factor Va. This latter value was determined from direct measurement of steady-state thrombin production. A comparable value of Kd = 1.0 +/- 0.1 pM was found by repeating these experiments in suspensions of phospholipid vesicles, and it was verified that prothrombinase assembly was not influenced by the addition of prothrombin. Using a minute amount (0.094 fmol cm-2) of preadsorbed factor Va, it was found that the rate of prothrombinase assembly exceeds the rate of collisions between Xa molecules from the buffer and the sparse Va molecules on the bilayer. Apparently, factor Xa adsorbs first to the membrane and then associates rapidly with factor Va by lateral diffusion. The data indicate almost instantaneous equilibrium of this complex formation on the surface with a lower limit for the bimolecular rate constant of kon = 2.8 x 10(13) (mol/cm2)-1 s-1. In suspensions of small phospholipid vesicles, prothrombinase assembly is collisionally limited and the value of kon should be proportional to vesicle diameter. This was verified with a method for estimation of kon values from thrombin generation curves. Values of 0.36 x 10(9) and 1.6 x 10(9) M-1 s-1 were found for vesicles of 20-30- and 60-80-nm diameter, respectively.     

Clustering of lipid-bound annexin V may explain its anticoagulant effect.

In 1985 we isolated a new vascular anticoagulant protein VAC alpha, now called annexin V, with a high binding affinity (Kd less than 10(-10) M) for phospholipids. Its anticoagulant effect was attributed to displacement of coagulation factors from the phospholipid membrane. The present study demonstrates that the inhibition of prothrombinase activity by annexin V strongly depends on the curvature of the membrane surface and on the calcium concentration. Half-maximal inhibition of prothrombinase on and binding of annexin V to small vesicles, composed of 20% phosphatidylserine and 80% phosphatidylcholine, requires 2-3 mM calcium. With large vesicles and planar bilayers considerably less calcium is required for inhibition of prothrombinase and for lipid binding. Half-maximal binding of annexin V to large vesicles and to planar bilayers occurs at 0.7 and 0.2 mM calcium, respectively. This seemingly confirms the displacement model. The displacement of coagulation factors, however, proved to be incomplete, with residual surface concentrations of factors Xa, Va, and prothrombin sufficient for effective production of thrombin. Cryoelectron microscopy revealed that annexin V binding to large vesicles caused planar facets, indicating the formation of large sheets of clustered annexin V. Apparently, the formation of these two-dimensional arrays is promoted by calcium and hampered by high surface curvature. It is speculated that the complete inhibition (greater than 99%) of prothrombinase activity by annexin V is caused by the reduced lateral mobility of prothrombin and factor Xa in rigid sheets of annexin V covering the membrane.     

Identification of variable simple sequence motifs in 19q13.2-qter: markers for the myotonic dystrophy locus.

Variable simple sequence motifs (VSSMs), or microsatellites, were used for the genetic delimitation of the myotonic dystrophy (DM) region at 19q. Three simple sequence motifs were identified in and around the ERCC1 DNA-repair gene at 19q13.2-13.3 and one in the vicinity of the RRAS gene at 19q13.3-qter. A (TG)n repeat, situated within the ninth intron of the ERCC1 gene, was converted into a highly informative multiallelic marker using PCR-mediated DNA amplification and high-resolution gel analysis. The structurally similar sequence motif in the RRAS gene yielded a marker system with only two alleles. Use of these VSSMs for linkage analysis and haplotyping in a selected set of DM families revealed that the DM gene is distal but close to the ERCC1 locus and can be excluded from the CKM-ERCC1 interval at 19q13.2. The order for RRAS and other distally located markers was established as DM-D19S50-[RRAS,KLK]-D19S22-ter.     

The effect of genomic information on optimal contribution selection in livestock breeding programs

Background

Long-term benefits in animal breeding programs require that increases in genetic merit be balanced with the need to maintain diversity (lost due to inbreeding). This can be achieved by using optimal contribution selection. The availability of high-density DNA marker information enables the incorporation of genomic data into optimal contribution selection but this raises the question about how this information affects the balance between genetic merit and diversity.

Methods

The effect of using genomic information in optimal contribution selection was examined based on simulated and real data on dairy bulls. We compared the genetic merit of selected animals at various levels of co-ancestry restrictions when using estimated breeding values based on parent average, genomic or progeny test information. Furthermore, we estimated the proportion of variation in estimated breeding values that is due to within-family differences.

Results

Optimal selection on genomic estimated breeding values increased genetic gain. Genetic merit was further increased using genomic rather than pedigree-based measures of co-ancestry under an inbreeding restriction policy. Using genomic instead of pedigree relationships to restrict inbreeding had a significant effect only when the population consisted of many large full-sib families; with a half-sib family structure, no difference was observed. In real data from dairy bulls, optimal contribution selection based on genomic estimated breeding values allowed for additional improvements in genetic merit at low to moderate inbreeding levels. Genomic estimated breeding values were more accurate and showed more within-family variation than parent average breeding values; for genomic estimated breeding values, 30 to 40% of the variation was due to within-family differences. Finally, there was no difference between constraining inbreeding via pedigree or genomic relationships in the real data.

Conclusions

The use of genomic estimated breeding values increased genetic gain in optimal contribution selection. Genomic estimated breeding values were more accurate and showed more within-family variation, which led to higher genetic gains for the same restriction on inbreeding. Using genomic relationships to restrict inbreeding provided no additional gain, except in the case of very large full-sib families.     

A new principle for rapid immunoassay of proteins based on in situ precipitate-enhanced ellipsometry

A new technique is presented that allows measurement of protein concentrations in the picomolar range with an assay time of only 10-20 min. The method is an enzyme-linked immunosorbent assay (ELISA), but uses in-situ ellipsometric measurement of a precipitating enzyme product instead of the usual colorimetric detection of accumulating enzyme product in solution. Quantitative validation was obtained by use of annexin V, a protein with high binding affinity for phosphatidylserine-containing phospholipid membranes, resulting in a transport-limited adsorption rate. This property was exploited to obtain a range of low surface concentrations of annexin V by timed exposures of phospholipid bilayers to known concentrations of annexin V. Using polyvinylchloride (PVC)-coated and silanized silicon slides, various versions of this technique were used for the rapid assay of fatty acid-binding protein (FABP), a recently introduced early marker for acute myocardial infarction with a normal plasma concentration below 1 nmol/l, interleukin 6 (IL-6), a cytokine with normal plasma concentrations below 1 pmol/l, and again, annexin V. A possible future application of the method in the development of a one-step ELISA is discussed.     

Analysis and test of laws for backward (metacontrast) masking

In backward visual masking, it is common to find that the mask has its biggest effect when it follows the target by several tens of milliseconds. Research in the 1960s and 1970s suggested that masking effects were best characterized by the stimulus onset asynchrony (SOA) between the target and mask. In particular, one claim has been that the SOA for which masking is optimal remains fixed, even as target and mask durations varied. Experimental evidence supported this claim, and it was accepted as an SOA law. However, recent modeling (Francis, 1997) and experimental studies (Macknik and Livingstone, 1998) argued for new ISI (interstimulus interval) and STA (stimulus termination asynchrony) laws, respectively. This paper reports a mathematical analysis and experimental tests of the laws. The mathematical analysis demonstrates unsuspected relationships between the laws. The experiments test the predictions of the SOA, ISI, and STA laws. The data favor the ISI law over the SOA and the STA laws.     

Influence of gravity compensation on muscle activity during reach and retrieval in healthy elderly

IntroductionArm support like gravity compensation may improve arm movements during stroke rehabilitation. It is unknown how gravity compensation affects muscle activation patterns during reach and retrieval movements. Since muscle activity during reach is represented by a component varying with movement velocity and a component supposedly counteracting gravity, we hypothesized that gravity compensation decreases the amplitude of muscle activity, but does not affect the pattern. To examine this, we compared muscle activity during well defined movements with and without gravity compensation in healthy elderly.MethodsTen subjects performed reach and retrieval movements with and without gravity compensation. Muscle activity of biceps, triceps, anterior, middle and posterior parts of deltoid and upper trapezius was compared between the two conditions.ResultsThe level of muscle activity was lower with gravity compensation in all muscles, reaching significance in biceps, anterior deltoid and trapezius (p ? 0.026). The muscle activation pattern did not differ between movements with and without gravity compensation (p ? 0.662).DiscussionGravity compensation only influenced the level of muscle activity but not the muscle activation pattern in terms of timing. Future studies should examine if the influence of gravity compensation is comparable for stroke patients. This may stimulate early and intensive training during rehabilitation.