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.     

Cross-linking behavior and foaming properties of bovine α-lactalbumin after glycation with various saccharides

α-Lactalbumin was glycated via the Maillard reaction in the dry state using various mono- and oligosaccharides. The reaction resulted not only in coupling of the saccharides to α-lactalbumin but also in cross-linked proteins. The glycation rate and the extent of cross-link formation were highly dependent on the saccharide used. Glycation by arabinose and xylose led to a very fast protein cross-link formation, whereas glucose showed a relatively low protein cross-linking ability. The stability of foams, created using the various glycated protein samples, depended on the type of saccharide used, the extent of glycation, and possibly the amount of cross-linked protein. Compared to nonmodified α-lactalbumin, glycation with rhamnose and fucose improved foam stability, whereas application of glucose, galacturonic acid, and their oligosaccharides did not exert a clear effect. Mass spectrometric analysis revealed that dehydration of the Amadori products is an indicator of the formation of protein cross-links.     

A scanning and transmission electron microscopic study of rotavirus-induced intestinal lesions in neonatal gnotobiotic dogs

Neonatal gnotobiotic dogs orally inoculated with canine rotavirus had ultrastructural changes limited to the jejunal and ileal regions of the small intestine. Early scanning electron microscopic findings consisted of swollen villus epithelial cells, denuded foci on intestinal villi, and slight to moderate villus atrophy. Later changes were slight villus atrophy with no denuded intestinal villi. Transmission electron microscopic changes in villus epithelial cells from 12 to 48 hours post-inoculation included: rotavirus particles associated with intracytoplasmic vacuoles near the terminal web and apical tubules; viral particles in dilated cisternae of rough endoplasmic reticulum; and moderate numbers of necrotic cells having no microvilli, swollen mitochondria, membrane-bound lipid-like material in the cytoplasm, clumped chromatin around the periphery of the nucleus, and disruption of the cytoplasmic membrane. In jejunum and ileum at 72 to 154 hours post-inoculation, there were fewer necrotic villus epithelial cells and fewer virus particles. In addition, the ultrastructural morphology of the majority of the villus epithelial cells was similar to crypt epithelium. These studies showed that rotavirus infected the villus epithelial cells with subsequent propagation of the rotavirus and destruction of villus epithelial cells.     

Inoculation of neonatal gnotobiotic dogs with a canine rotavirus

Neonatal gnotobiotic dogs were inoculated orally with a rotavirus isolated from a pup with fatal diarrhea, and in the gnotobiotic dogs, diarrhea was observed between postinoculation hours (PIH) 20 and 24. The diarrhea persisted through PIH 154, and inoculated pups had clinical signs of dehydration after PIH 24. Negative-contrast electron microscopy of the feces from inoculated pups revealed rotavirus particles from PIH 12 through 154. Using an indirect-fluorescent antibody test, serum rotavirus antibody was detected in inoculated pups by PIH 96. In the duodenum, jejunum, and ileum of inoculated pups, group-specific rotaviral antigen was observed within absorptive villus epithelial cells and mononuclear cells in the villus lamina propria with an indirect-fluorescent antibody test. Fluorescence was seen in the small intestine of inoculated pups killed by PIH 12 and was present in intestines of pups killed through PIH 154. Rotaviral antigen was also seen in the mesenteric lymph nodes of a few inoculated pups killed at PIH 48.     

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.     

Latitudinal variability in the seroprevalence of antibodies against Toxoplasma gondii in non-migrant and Arctic migratory geese

Toxoplasma gondii is an intracellular coccidian parasite found worldwide and is known to infect virtually all warm-blooded animals. It requires a cat (family Felidae) to complete its full life cycle. Despite the absence of wild felids on the Arctic archipelago of Svalbard, T. gondii has been found in resident predators such as the arctic fox and polar bear. It has therefore been suggested that T. gondii may enter this ecosystem via migratory birds. The objective of this study was to identify locations where goose populations may become infected with T. gondii, and to investigate the dynamics of T. gondii specific antibodies. Single blood samples of both adults and juveniles were collected from selected goose species (Anser anser, A. brachyrhynchus, Branta canadensis, B. leucopsis) at Arctic brood-rearing areas in Russia and on Svalbard, and temperate wintering grounds in the Netherlands and Denmark (migratory populations) as well as temperate brood-rearing grounds (the Netherlands, non-migratory populations). A modified agglutination test was used on serum, for detection of antibodies against T. gondii. Occasional repeated annual sampling of individual adults was performed to determine the antibody dynamics. Adults were found seropositive at all locations (Arctic and temperate, brood-rearing and wintering grounds) with low seroprevalence in brood-rearing birds on temperate grounds. As no juvenile geese were found seropositive at any brood-rearing location, but nine month old geese were found seropositive during spring migration we conclude that geese, irrespective of species and migration, encounter T. gondii infection in wintering areas. In re-sampled birds on Svalbard significant seroreversion was observed, with 42% of seropositive adults showing no detectable antibodies after 12 months, while the proportion of seroconversion was only 3%. Modelled variation of seroprevalence with field data on antibody longevity and parasite transmission suggests seroprevalence of a population within a range of 5.2–19.9%, in line with measured values. The high occurrence of seroreversion compared to the low occurrence of seroconversion hampers analysis of species- or site-specific patterns, but explains the absence of an increase in seroprevalence with age and the observed variation in antibody titre. These findings imply that even though infection rate is low, adults introduce T. gondii to the high Arctic ecosystem following infection in temperate regions.     

Community patterns of soil bacteria and nematodes in relation to geographic distance

Ecosystems consist of aboveground and belowground subsystems and the structure of their communities is known to change with distance. However, most of this knowledge originates from visible, aboveground components, whereas relatively little is known about how soil community structure varies with distance and if this variability depends on the group of organisms considered. In the present study, we analyzed 30 grasslands from three neighboring chalk hill ridges in southern UK to determine the effect of geographic distance (1–198 km) on the similarity of bacterial communities and of nematode communities in the soil. We found that for both groups, community similarity decayed with distance and that this spatial pattern was not related to changes either in plant community composition or soil chemistry. Site history may have contributed to the observed pattern in the case of nematodes, since the distance effect depended on the presence of different nematode taxa at one of the hill ridges. On the other hand, site-related differences in bacterial community composition alone could not explain the spatial turnover, suggesting that other factors, such as biotic gradients and local dispersal processes that we did not include in our analysis, may be involved in the observed pattern. We conclude that, independently of the variety of causal factors that may be involved, the decay in similarity with geographic distance is a characteristic feature of both communities of soil bacteria and nematodes.     

Seeds capture the diversity of genetic resource collections of <Emphasis Type="Italic">Malus sieversii</Emphasis> maintained in an orchard

Many species in genebank collections are maintained vegetatively either as in vitro cultures or as plants in the field or greenhouse. In these cases, individual genotypes (cultivars) are the focus of conservation. In crop wild relatives, where the value of an accession is in the heterogeneity of segregating genotypes, seeds may be the most representative form of conservation. We used two sets of field-planted Malus sieversii apple trees that originated from seeds collected from two wild populations in Kazakhstan as a model system to determine if seeds from hand-pollinated crosses could be used to represent the allelic diversity of the parent trees in a genebank orchard. A crossing design was developed that used each of the selected trees from the two Kazakhstan collection sites as mothers and also as fathers in the form of bulked pollen pools. The sets of offspring from these crosses were genotyped using microsatellite markers. Paternity analyses revealed the percentage of offspring that was derived from each of the parent trees. We also determined the contribution of each parent to the fertilization events from the pollen pools. Although there were some unintended pollination events (fertilization by trees in unexpected pollen pools) and unequal contribution of pollen parents within pollen pools, we demonstrated that the seedlings effectively captured the allelic frequency and diversity of the parents. A seed collection strategy is the most efficient way to conserve the diversity of crop wild relatives even in collections typically conserved as individual clones.