Peroxisomal Degradation of 2-Oxoisocaproate. Evidence for Free Acid Intermediates

Peroxisomes from mung bean hypocotyl (Vigna radiata L.) degrade 2-oxoisocaproate, the transamination product of leucine, via isobutyryl-CoA and propionyl-CoA to acetyl-CoA. The methyl group at the C-3 position forms a barrier to β-oxidation. This barrier is overcome in the peroxisomes by several enzymatic steps. Senecioate (3-methylcrotonate), 2-hydroxyisovalerate, and 2-oxoisovalerate were detected as free acid intermediates. Senecioate, formed from 3-methylcrotonyl-CoA, is transformed by enzymatic hydrolysis to 2-hydroxyisovalerate. 2-Hydroxyisovalerate is then oxidized to 2-oxoisovalerate in an H₂O₂-producing reaction, exhibiting 1:1 stoichiometry of the products, by a 2-hydroxyacid oxidase which is different from the peroxisomal marker enzyme glycollate oxidase. 2-oxoisovalerate is activated by an NAD-dependent oxidative decarboxylation to isobutyryl-CoA. Accumulation of 2-oxoisovalerate in the presence of arsenite, an inhibitor of oxidative decarboxylations, is a feature of this latter pathway of degradation of isovaleryl-CoA or senecioate. It is concluded that the barrier caused by the methyl group of 2-oxoisocaproate is surmounted in higher plant peroxisomes in a manner different to that in mammalian mitochondria.     

Snap,crackle, and pop: Acoustic-based model estimation of snapping shrimp populations in healthy and degraded hard-bottom habitats

Human use of the ocean and its ecosystems continues to degrade coastal habitats around the world. Assessing anthropogenic impacts on these environments can be costly and manpower-intensive; thus, the development of rapid, remote techniques to assess habitat quality is important. We employed autonomous hydrophone receivers to record the soundscapes of healthy, sponge-rich hard-bottom habitat in Florida Bay, Florida (USA) and hard-bottom areas impacted by sponge die-offs. We also recorded sounds emanating from individual sponges of three species that were isolated in underwater sound booths, and then enumerated the invertebrates (mostly snapping shrimp) dwelling within the canals of each sponge. From these recordings, a modified cylindrical sound propagation model was used to estimate distances to individual snapping shrimp snaps. Using the program Distance, we estimated snapping shrimp population density and abundance within both habitat types. More snapping shrimp snaps per unit time were recorded in healthy hard-bottom areas as compared to degraded hard-bottom areas. In addition, the average distance to a snap source was greater within degraded hard-bottom areas than within healthy hard-bottom areas. As a consequence, the estimated density and abundance of snapping shrimp were one to two orders of magnitude greater within healthy habitat than within degraded habitat. This study demonstrates the feasibility of using acoustic sampling and modeling to rapidly assess populations of soniferous benthic indicator species, whose vocalizations may yield indirect estimates of habitat quality.     

Protein Turnover in Brain of the Rat Fetus

Protein synthesis in vivo was studied in whole brain of rat fetuses using continuous intravenous infusion of L-[U-14C]tyrosine into unrestrained pregnant rats at 19 and 21 days gestation. Protein degradation (KD) was calculated by subtracting fractional growth rate of brain protein (KG) from the fractional synthesis rate (KS). KS was high at both gestational ages (0.42 +/- 0.03 days-1 at day 19, 0.47 +/- 0.029 days-1 at 21 days), comparable to values previously reported for newborn rat cerebral hemispheres, and threefold higher than is seen in adult animals. KD was similar at both 19 and 21 days gestation (0.19-0.24) and lower than that reported in neonatal rat brain using similar techniques. Protein accretion during the most rapid phase of brain growth (fetus) is accomplished by similar rates of protein synthesis, but decreased rates of degradation when compared with a slower growth phase (newborn). KD in the brain of the rapidly growing fetus is slightly higher than in adult cerebral hemispheres.     

Protein disulphide isomerase is required for signal peptide peptidase-mediated protein degradation

The human cytomegalovirus glycoprotein US2 induces dislocation of MHC class I heavy chains from the endoplasmic reticulum (ER) into the cytosol and targets them for proteasomal degradation. Signal peptide peptidase (SPP) has been shown to be integral for US2-induced dislocation of MHC class I heavy chains although its mechanism of action remains poorly understood. Here, we show that knockdown of protein disulphide isomerase (PDI) by RNA-mediated interference inhibited the degradation of MHC class I molecules catalysed by US2 but not by its functional homolog US11. Overexpression of the substrate-binding mutant of PDI, but not the catalytically inactive mutant, dominant-negatively inhibited US2-mediated dislocation of MHC class I molecules by preventing their release from US2. Furthermore, PDI associated with SPP independently of US2 and knockdown of PDI inhibited SPP-mediated degradation of CD3δ but not Derlin-1-dependent degradation of CFTR DeltaF508. Together, our data suggest that PDI is a component of the SPP-mediated ER-associated degradation machinery.     

In vitro protein digestion kinetics of protein sources for pigs

In current feed evaluation systems, the nutritional value of protein sources in diets for pigs is based on the ileal digestibility of protein and amino acids, which does not account for the kinetics of protein digestion along the gastrointestinal tract. The objective of the present study was to determine the in vitro protein digestion kinetics of different protein sources (soya bean meal (SBM), wheat gluten (WG), rapeseed meal (RSM), whey powder (WP), dried porcine plasma protein, yellow meal worm larvae and black soldier fly larvae (BSF)). Protein sources were incubated with pepsin at pH 3.5 for 0 to 90 min and subsequently with pancreatin at pH 6.8 for 0 to 210 min at 39°C. The in vitro protein digestion kinetics were described as the kinetics of nitrogen (N) solubilisation and the release of low molecular weight peptides (LMW) (<500 Da). The N solubilisation rate ranged from 0.025 min⁻¹ for BSF to 0.685 min⁻¹ for WP during the incubation with pepsin, and from 0.027 min⁻¹ for RSM to 0.343 min⁻¹ for WP during the incubation with pancreatin. The release rate of LMW peptides ranged from 0.027 min⁻¹ for WG to 0.093 min⁻¹ for WP during the incubation with pepsin, and from 0.029 min⁻¹ for SBM to 0.385 min⁻¹ for WP. Black soldier fly larvae showed a similar release rate of LMW peptides as WP during the incubation with pancreatin. At the end of the sequential incubation with pepsin (90 min) and pancreatin (210 min), WG and WP showed the highest percentage of N present in LMW peptides relative to total N (78% and 79%, respectively), whereas SBM showed the lowest (35%). In conclusion, protein sources for pig diets show substantial differences in in vitro protein digestion kinetics as measured by the kinetics of N solubilisation and the release of LMW peptides. The rate of release of LMW peptides was not correlated to the rate of N solubilisation for each of the protein sources evaluated.     

Exophiala macquariensis sp. nov., a cold adapted black yeast species recovered from a hydrocarbon contaminated sub-Antarctic soil

A new black yeast species, Exophiala macquariensis is described that is a member of the ascomycete family Herpotrichiellaceae, order Chaetothyriales. The genus Exophiala is comprised of opportunistic pathogens isolated from clinical specimens as well as species recovered from hydrocarbon contaminated environments. Several species have been reported to be able to degrade benzene, toluene, ethylbenzene and xylenes. Here, a novel species of Exophiala (CZ06) previously isolated from a Sub-Antarctic, Macquarie Island soil that was spiked with Special Antarctic Blend diesel fuel (SAB) is described. This isolate has the capacity of toluene biodegradation at cold temperatures. Multilocus sequence typing showed that this fungus was closely related to the pathogenic species Exophiala salmonis and Exophiala equina. With the capacity to utilise hydrocarbons as a sole carbon source at 10 °C, this fungus has great potential for future bioremediation applications.