Identification of the peroxidase-generated intermolecular dityrosine cross-link in bovine α-lactalbumin

The peroxidase-mediated oxidation of calcium-depleted bovine α-lactalbumin generates a mixture of covalently bound protein oligomers with interesting foaming properties. Here, we isolated the initially formed covalent α-lactalbumin dimer and studied its mode of cross-linking. Liquid chromatography-Fourier transform mass spectrometry (LC-FTMS) of proteolytic digests revealed the unambiguous identification of a peroxidase-catalyzed covalent link between Tyr18 and Tyr50. This shows that, although the radical reaction is often regarded as a random reaction, the initial product formation is specific. Protein structural modeling indicates that the conjugation reaction between these tyrosines is sterically favored and involves initial noncovalent protein complex formation through charge compensation, facilitating intermolecular cross-linking. The identification of the Tyr18-Tyr50 cross-link supports the view that the peroxidase-mediated oxidation of apo α-lactalbumin is a sequential process, involving the formation of linear trimers and higher order oligomers.     

Erfl?rung, betressend die allgemeine National?fonomie, herausgegeben von Dr. Leo (Underlind)

Ohne Zusammenfassung     

Prevalence of pigment gallstones in sheep.

In a survey of 666 sheep at a slaughterhouse, gallstones (concretions with a diameter greater than or equal to 1 mm) were found in the gallbladder of 50 sheep (7.5%), sludge (concretions with a diameter less than 1 mm) was found in 9 sheep (1.4%), and sludge plus gallstones were found in 7 sheep (1.1%). Gallstones and sludge were associated, and were more frequent in lambs and females, compared with adults and males. Qualitative analysis of the stones revealed all to be pigment (bilirubin) stones. There was a statistically significant increase of biliary bilirubin (total and indirect quota) only in sheep with gallstones plus sludge, compared with control sheep without sludge or gallstones. Concentrations of bilirubin, cholesterol, phospholipids, total and single bile aids, and total and ionized calcium were similar in the bile of sheep with gallstones, sludge, or both and control sheep. Bacteriologic analysis of the bile in 10 sheep with gallstones and 10 controls revealed bacteria in 50% of the first group and in 75% of the second group (Escherichia coli in all sheep and Salmonella spp also in 1 sheep with gallstones). These findings confirm our earlier findings of a high prevalence of black pigment gallstones in sheep. On that basis, we suggest that gallstones are associated with high total bilirubin concentration in the bile, and deconjugating bacteria are common in the biliary tract of these animals.     

Biochar and fertiliser applications influence phosphorus fractionation and wheat yield

The use of biochar in agriculture to achieve the dual benefits of improving soil quality whilst sequestering carbon (C) has received much attention. However, in low-intensity broadacre agricultural systems where yield is constrained by rainfall and costs associated with phosphorus (P) fertiliser, the application of biochar at rates commonly reported (>10 t ha^?1) are likely to be prohibitively expensive where yield benefits cannot be guaranteed. In marginal areas where calcareous soils dominate, biochar application has no liming effect, reducing its value compared to application in acidic soils. In the present study, we use a field experiment to investigate the interaction between P fertilisation and biochar banding at low application rates (<1 t ha^?1) on wheat yield and soil P fractionation (assessed by a modified Hedley method) in a highly alkaline Haplic Calcisol in a dryland broadacre cropping system. Our results demonstrate no statistically significant effect of low rate biochar banding on wheat yield in this highly P-constrained soil, but a significant effect of both biochar and fertiliser on P fractionation in both years of the study. Higher P fertiliser rates significantly increased wheat yield in all biochar treatments. The interactions between biochar, P fertiliser and P fractionation indicate shifts in potential P availability both as a result of P fertilisation and also biochar application. Further work is required in low productivity calcareous systems such as that studied here to elucidate the potential for biochar amendment to improve productivity and sequester C.     

Eye-specific retinogeniculate segregation independent of normal neuronal activity

The segregation of initially intermingled left and right eye inputs to the dorsal lateral geniculate nucleus (DLGN) during development is thought to be in response to precise spatial and temporal patterns of spontaneous ganglion cell activity. To test this hypothesis, we disrupted the correlated activity of neighboring ganglion cells in the developing ferret retina through immunotoxin depletion of starburst amacrine cells. Despite the absence of this type of correlated activity, left and right eye inputs segregated normally in the DLGN. By contrast, when all spontaneous activity was blocked, the projections from the two eyes remained intermingled. Thus, certain features of normal neural activity patterns are not required for the formation of eye-specific projections to the DLGN.     

Haploid females in the parasitic wasp Nasonia vitripennis

The insect order of Hymenoptera (ants, bees, sawflies, and wasps) consists almost entirely of haplodiploid species. Under haplodiploidy, males develop from unfertilized eggs and are haploid, whereas females develop from fertilized eggs and are diploid. Although diploid males commonly occur, haploid females have never been reported. In analyzing the phenomenon of gynandromorphism in the parasitoid wasp Nasonia vitripennis, we found a line that generates complete phenotypic females from unfertilized eggs. These females have ovaries, can lay eggs, and are haploid, as shown by cytological and flow cytometric analyses. The data show that diploidy is not necessary for female development.     

Kinetic study of flavonoid reactions with stable radicals

The antiradical activities of some flavonols (kaempferol, quercetin, robinetin, quercetagetin, and myricetin), flavones (apigenin, baicalein, and luteolin), flavanones (naringenin and dihydroquercetin), and flavanols [(+)-catechin and (-)-epicatechin] were determined by measuring the reaction kinetics with 2,2-diphenyl-1-picrylhydrazyl (DPPH) and alpha,gamma-bisdiphenylene-beta-phenylallyl (BDPA) radicals. The reactions, which follow the mixed second-order rate law, were investigated under pseudo-first-order conditions by use of a large excess of flavonoids, and their stoichiometry was determined by spectrophotometric titration. The results confirm stoichiometric factors of 1, 2, and 3 for flavonoids with one, two, and three hydroxyl groups in the B-ring, respectively, excluding kaempferol, which, despite a single OH group in the B-ring, has a factor of 2, which is explained by the 3-OH group supporting the reaction with free radicals. Structure-activity considerations indicate for the present series of flavonoids the importance of multiple OH substitutions and conjugation. The logarithms of reaction rate constants with the OH, DPPH, and BDPA radicals correlate well with the reduction potential of the flavonoids.     

Modification of glutamine and lysine residues in holo and apo alpha-lactalbumin with microbial transglutaminase

The molecular structures determine the physical properties of milk proteins and are important for the texture of many dairy-based foods. Bovine alpha-lactalbumin (alpha-LA) is a globular 123 amino acid Ca(2+) binding milk protein. Modification with microbial Ca(2+) independent transglutaminase (MTGase) was used to modify lysines and glutamines in holo and apo alpha-LA. At 30 degrees C no lysines or glutamines are modified in holo alpha-LA, whereas in apo alpha-LA lysines 13, 16, 108, and 114, and glutamines 39 and 43, are modified. At 50 degrees C lysines 13, 16, 108, and 114, but no glutamines, are modified in holo alpha-LA, whereas in apo alpha-LA lysines 5, 13, 16, 108, and 114, and glutamines 39, 43, 54, 65, and 117, are modified. The methods presented here offer the possibility to manipulate the availabilities of residues in alpha-LA to the MTGase reaction and enable the preparation of alpha-LA species with different degrees of modification and hence with different physical properties.