Estimation of genetic parameters of boar taint; skatole and androstenone and their correlations with sexual maturation

Boar taint is mainly caused by two components; skatole (3-methylindole) and androstenone. By castrating the male pigs, boar taint will be avoided. In Norway, castration of pigs will no longer be permitted after 2009. This represents a substantial cost for the Norwegian swine production. Other Norwegian studies have shown that a large proportion of pigs are above the consumer detection limits for these two chemical components. The obvious question for the geneticist arises: Is it possible to select against skatole and androstenone in a breeding programme? Skatole is produced in the gut by bacteria. It is then absorbed in the blood stream. Skatole is either metabolised in the liver or transported and stored in fatty tissue. Androstenone is produced in the testis, and its biochemical pathway is related to the pathway of testosterone. In this study, fatty tissue was collected from the carcasses of Norwegian Landrace and Duroc boars, and analysed for androstenone and skatole. The length of glandula bulbourethralis was measured on the same animals, as this is regarded as a good indicator of sexual maturation. Heritabilities of androstenone and skatole were substantial. The two components were genetically correlated. Sexual maturation was also highly heritable. However, correlations to both androstenone and skatole were significantly unfavourable.     

Brown shrimp (Crangon crangon, L.) functional response to density of different sized juvenile bivalves Macoma balthica (L.)

Variability in infaunal bivalve abundance in the Wadden Sea is largely determined by recruitment variability. Post-settlement, but pre-recruitment bivalve mortality is high and related to the occurrence of their most abundant predator, the brown shrimp Crangon crangon. To investigate if the mortality patterns of newly settled bivalves can be explained by the foraging behavior of brown shrimp, we carried out experiments on shrimp functional response to three size classes of juveniles of the Baltic Tellin Macoma balthica. The functional response curves for all three prey sizes (0.62 mm, 0.73 mm, and 0.85 mm) were the hyperbolic Holling's type II. The attack rate was highest for the smallest prey size (a = 0.31, medium and large prey a = 0.22); the handling time was longest for the largest prey size (T_h = 29 s, small and medium prey T_h = 15 s). Thus, a large body size is advantageous for the bivalves over the whole density range. Knowledge of individual foraging behavior is needed to model predation mortality of bivalves. The consumption rates in the experiment were theoretically high enough to account for M. balthica mortality in the field.     

Short/branched-chain acyl-CoA dehydrogenase deficiency due to an IVS3+3A>G mutation that causes exon skipping

Short/branched-chain acyl-CoA dehydrogenase deficiency (SBCADD) is an autosomal recessive disorder of l-isoleucine catabolism. Little is known about the clinical presentation associated with this enzyme defect, as it has been reported in only a limited number of patients. Because the presence of C5-carnitine in blood may indicate SBCADD, the disorder may be detected by MS/MS-based routine newborn screening. It is, therefore, important to gain more knowledge about the clinical presentation and the mutational spectrum of SBCADD. In the present study, we have studied two unrelated families with SBCADD, both with seizures and psychomotor delay as the main clinical features. One family illustrates the fact that affected individuals may also remain asymptomatic. In addition, the normal level of newborn blood spot C5-acylcarnitine in one patient underscores the fact that newborn screening by MS/MS currently lacks sensitivity in detecting SBCADD. Until now, seven mutations in the SBCAD gene have been reported, but only three have been tested experimentally. Here, we identify and characterize an IVS3+3A>G mutation (c.303+3A>G) in the SBCAD gene, and provide evidence that this mutation is disease-causing in both families. Using a minigene approach, we show that the IVS3+3A>G mutation causes exon 3 skipping, despite the fact that it does not appear to disrupt the consensus sequence of the 5′ splice site. Based on these results and numerous literature examples, we suggest that this type of mutation (IVS+3A>G) induces missplicing only when in the context of non-consensus (weak) 5′ splice sites. Statistical analysis of the sequences shows that the wild-type versions of 5′ splice sites in which +3A>G mutations cause exon skipping and disease are weaker on average than a random set of 5′ splice sites. This finding is relevant to the interpretation of the functional consequences of this type of mutation in other disease genes.     

Anammox enrichment from reject water on blank biofilm carriers and carriers containing nitrifying biomass: operation of two moving bed biofilm reactors (MBBR)

The anammox bacteria were enriched from reject water of anaerobic digestion of municipal wastewater sludge onto moving bed biofilm reactor (MBBR) system carriers-the ones initially containing no biomass (MBBR1) as well as the ones containing nitrifying biomass (MBBR2). Duration of start-up periods of the both reactors was similar (about 100?days), but stable total nitrogen (TN) removal efficiency occurred earlier in the system containing nitrifying biomass. Anammox TN removal efficiency of 70% was achieved by 180?days in both 20?l volume reactors at moderate temperature of 26.0°C. During the steady state phase of operation of MBBRs the average TN removal efficiencies and maximum TN removal rates in MBBR1 were 80% (1,000?g-N/m(3)/day, achieved by 308?days) and in MBBR2 85% (1,100?g-N/m(3)/day, achieved by 266?days). In both reactors mixed bacterial cultures were detected. Uncultured Planctomycetales bacterium clone P4, Candidatus Nitrospira defluvii and uncultured Nitrospira sp. clone 53 were identified by PCR-DGGE from the system initially containing blank biofilm carriers as well as from the nitrifying biofilm system; from the latter in addition to these also uncultured ammonium oxidizing bacterium clone W1 and Nitrospira sp. clone S1-62 were detected. FISH analysis revealed that anammox microorganisms were located in clusters in the biofilm. Using previously grown nitrifying biofilm matrix for anammox enrichment has some benefits over starting up the process from zero, such as less time for enrichment and protection against severe inhibitions in case of high substrate loading rates.     

Paired assessment of volatile anesthetic concentrations with synaptic actions recorded in vitro

The volatile anesthetic isoflurane poses a number of experimental challenges in the laboratory. Due to its rapid evaporation, the open conditions of most in vitro electrophysiological recording systems make the determination of actual isoflurane concentrations a challenge. Since the absolute anesthetic concentration in solution is directly related to efficacy, concentration measurements are important to allow comparisons between laboratory and clinical studies. In this study we quantify the sources of isoflurane loss during experimentation and describe a method for the measurement of isoflurane concentrations using gas chromatography and mass spectrometry simultaneous to in vitro electrophysiological measurements. Serial samples of perfused bath solution allowed correlation of isoflurane concentrations with ongoing biological effects. Saturated physiological solutions contained 13.4 +/- 0.2 mM isoflurane and were diluted to desired "nominal" concentrations for experiments. The perfusion system established stable isoflurane concentrations within the bath by 2 minutes. However, bath isoflurane concentrations varied substantially and unpredictably between experiments. The magnitudes of such discrepancies in isoflurane concentrations spanned clinically important levels. Our studies suggest that, despite countermeasures, solution handling significantly impacted the isoflurane content in the tissue bath. The magnitude of these discrepancies appears to necessitate systematic direct measurement of bath isoflurane concentrations during most in vitro conditions.     

Molecular basis of phospholipase A2 activity toward phospholipids with sn-1 substitutions

We studied secretory phospholipase A₂ type IIA (sPLA₂) activity toward phospholipids that are derivatized in the sn-1 position of the glycerol backbone. We explored what type of side group (small versus bulky groups, hydrophobic versus polar groups) can be introduced at the sn-1 position of the glycerol backbone of glycerophospholipids and at the same time be hydrolyzed by sPLA₂. The biophysical characterization revealed that the modified phospholipids can form multilamellar vesicles, and several of the synthesized sn-1 functionalized phospholipids were hydrolyzed by sPLA₂. Molecular dynamics simulations provided detailed insight on an atomic level that can explain the observed sPLA₂ activity toward the different phospholipid analogs. The simulations revealed that, depending on the nature of the side chain located at the sn-1 position, the group may interfere with an incoming water molecule that acts as the nucleophile in the enzymatic reaction. The simulation results are in agreement with the experimentally observed sPLA₂ activity toward the different phospholipid analogs.     

The Y42H mutation in medium-chain acyl-CoA dehydrogenase, which is prevalent in babies identified by MS/MS-based newborn screening, is temperature sensitive.

Medium-chain acyl-CoA dehydrogenase (MCAD) is a homotetrameric flavoprotein which catalyses the initial step of the beta-oxidation of medium-chain fatty acids. Mutations in MCAD may cause disease in humans. A Y42H mutation is frequently found in babies identified by newborn screening with MS/MS, yet there are no reports of patients presenting clinically with this mutation. As a basis for judging its potential consequences we have examined the protein phenotype of the Y42H mutation and the common disease-associated K304E mutation. Our studies of the intracellular biogenesis of the variant proteins at different temperatures in isolated mitochondria after in vitro translation, together with studies of cultured patient cells, indicated that steady-state levels of the Y42H variant in comparison to wild-type were decreased at higher temperature though to a lesser extent than for the K304E variant. To distinguish between effects of temperature on folding/assembly and the stability of the native enzyme, the thermal stability of the variant proteins was studied after expression and purification by dye affinity chromatography. This showed that, compared with the wild-type enzyme, the thermostability of the Y42H variant was decreased, but not to the same degree as that of the K304E variant. Substrate binding, interaction with the natural electron acceptor, and the binding of the prosthetic group, FAD, were only slightly affected by the Y42H mutation. Our study suggests that Y42H is a temperature sensitive mutation, which is mild at low temperatures, but may have deleterious effects at increased temperatures.     

Biogenesis of processing-competent secretory organelles in vitro.

Propeptide processing occurs in specific compartments of the secretory pathway, but how these processing-competent organelles are generated from their processing-incompetent precursor compartments is unknown. To dissect the process biochemically, we have developed a novel cell-free system reconstituting the production of processing-competent secretory granules in AtT-20 cells. Using donor membranes containing [(35)S]sulfate labeled pro-opiomelanocortin (POMC)(5) in the trans-Golgi, we can reconstitute cytosol- and ATP-dependent prohormone processing as well as incorporation of processed ACTH into immature secretory granules (ISGs). Under limiting cytosol conditions, both reactions are greatly stimulated by ADP-ribosylation factor 1 (ARF1) but not by the GDP-bound ARF1 T31N mutant. pH studies show that lumenal acidification, most likely due to ARF-mediated sorting of proton pumps and leaks during budding, confers processing competency to the resulting organelle. Surprisingly, comparison of onset of processing and ISG release reveals that they are distinct biochemical processes with different kinetics and separate cytosolic requirements. Moreover, ARF regulates the onset of prohormone processing but not ISG release. Our data suggest a two-step mechanism (onset of processing followed by ISG release) for the production of processing-competent organelles from the trans-Golgi and provide the first system with which these two steps may be individually dissected.