N-Linked glycosylation of a baculovirus-expressed recombinant glycoprotein in insect larvae and tissue culture cells

The potential of insect cell cultures and larvae infected with recombinant baculoviruses to produce authentic recombinant glycoproteins cloned from mammalian sources was investigated. A comparison was made of the N-linked glycans attached to secreted alkaline phosphatase (SEAP) produced in four species of insect larvae and their derived cell lines plus one additional insect cell line and larvae of one additional species. These data survey N-linked oligosaccharides produced in four families and six genera of the order Lepidoptera. Recombinant SEAP expressed by recombinant isolates of Autographa californica and Bombyx mori nucleopolyhedroviruses was purified from cell culture medium, larval hemolymph or larval homogenates by phosphate affinity chromatography. The N-linked oligosaccharides were released with PNGase-F, labeled with 8- aminonaphthalene-1-3-6-trisulfonic acid, fractionated by polyacrylamide gel electrophoresis, and analyzed by fluorescence imaging. The oligosaccharide structures were confirmed with exoglycosidase digestions. Recombinant SEAP produced in cell lines of Lymantria dispar (IPLB-LdEIta), Heliothis virescens (IPLB-HvT1), and Bombyx mori (BmN) and larvae of Spodoptera frugiperda, Trichoplusia ni , H.virescens , B.mori , and Danaus plexippus contained oligosaccharides that were structurally identical to the 10 oligosaccharides attached to SEAP produced in T.ni cell lines. The oligosaccharide structures were all mannose-terminated. Structures containing two or three mannose residues, with and without core fucosylation, constituted more than 75% of the oligosaccharides from the cell culture and larval samples.      

Tissue Level Compartmentation of (R)-Amygdalin and Amygdalin Hydrolase Prevents Large-Scale Cyanogenesis in Undamaged Prunus Seeds

Plum (Prunus domestica) seeds, which contain the cyanogenic diglucoside (R)-amygdalin and lesser amounts of the corresponding monoglucoside (R)-prunasin, release the respiratory toxin HCN upon tissue disruption. Amygdalin hydrolase (AH) and prunasin hydrolase (PH), two specific [beta]-glucosidases responsible for hydrolysis of these glucosides, were purified to near homogeneity by concanavalin A-Sepharose 4B and carboxymethyl-cellulose chromatography. Both proteins appear as polypeptides with molecular masses of 60 kD upon sodium dodecyl sulfate-polyacrylamide gel electrophoresis, but they exhibit different isoelectric points (PH, 5.6-6.0; AH, 7.8-8.2). AH and PH were localized within mature plum seeds by tissue printing, histochemistry, and silver-enhanced immunogold labeling. As was previously shown in black cherry (Prunus serotina) seeds (E.Swain, C.P. Li, J.E. Poulton [1992] Plant Physiol 100: 291-300), AH and PH are restricted to protein bodies of specific procambial cells and are absent from the cotyledonary parenchyma, bundle sheath, and endosperm cells. In contrast, the cyanogenic glycosides in both plum and black cherry seeds, which were detected by tissue printing, occur solely in the cotyledonary parenchyma and are absent from the procambium and endosperm. It is concluded that tissue level compartmentation prevents large-scale cyanoglycoside hydrolysis in intact Prunus seeds.     

Increased Biodiversity in the Environment Improves the Humoral Response of Rats

Previous studies have compared the immune systems of wild and of laboratory rodents in an effort to determine how laboratory rodents differ from their naturally occurring relatives. This comparison serves as an indicator of what sorts of changes might exist between modern humans living in Western culture compared to our hunter-gatherer ancestors. However, immunological experiments on wild-caught animals are difficult and potentially confounded by increased levels of stress in the captive animals. In this study, the humoral immune responses of laboratory rats in a traditional laboratory environment and in an environment with enriched biodiversity were examined following immunization with a panel of antigens. Biodiversity enrichment included colonization of the laboratory animals with helminths and co-housing the laboratory animals with wild-caught rats. Increased biodiversity did not apparently affect the IgE response to peanut antigens following immunization with those antigens. However, animals housed in the enriched biodiversity setting demonstrated an increased mean humoral response to T-independent and T-dependent antigens and increased levels of “natural” antibodies directed at a xenogeneic protein and at an autologous tissue extract that were not used as immunogens.     

Cell-cell communication and axis specification in the Drosophila oocyte

Intercellular communication between the somatic and germline cells is vital to development of the Drosophila egg chamber. One critical outcome of this communication is the polarization of the oocyte along the anterior-posterior axis, a process induced by an unknown signal from the somatic follicle cells to the oocyte. The existence of this signal has been inferred from several reports demonstrating that the differentiation and patterning of the follicle cells by the spatially restricted activation of certain cell-signaling pathways is necessary for axis formation in the oocyte. These reports have also provided a framework for understanding how these signaling pathways are integrated to generate the follicle-cell pattern, but the precise role of the follicle cells in anterior-posterior axis formation remains enigmatic. Research has identified several genes that appear to be involved in the polarizing communication from the follicle cells to the oocyte. Interestingly the proteins encoded by most of these genes are associated with the extracellular matrix, suggesting a pivotal role for this complex biological component in the polarizing communication between the follicle cells and the oocyte. This review summarizes the findings in this area, and uses the experimental analyses of these genes to evaluate various models describing the possible nature of the polarizing signal, and the role of these genes in it.     

Parsimonious modelling of nutrient fluxes for a terrestrial ecosystem on Svalbard

MBL-MEL, a simple model of ecosystem biogeochemistry, is amended and applied to plant and soil C, ¹⁴N and ¹⁵N data for the summers of 2001–2003 from Brandalpynten, a maritime high Arctic site on Svalbard following the application of ¹⁵N (99 atom%) as ¹⁵NO₃-N at or ¹⁵NH₄-N at concentrations of 1 or 5 kg N ha⁻¹. Variants of this Parent model are also developed to incorporate: temperature dependence into equations describing nutrient fluxes (Temp model); cryptogams (Cryp model); both features combined (CrypTemp model). Goodness-of-fit (GOF) statistics suggest that the addition of temperature-dependence improves the utility of models with and without cryptogams: the residual weighted sums of squares per data point were 5.69, 3.91, 4.31 and 3.93 for the Parent, Temp, Cryp and CrypTemp models respectively. The application of model selection criteria confirm that the addition of temperature-dependence also improves model generalisability. Across all models, the principal discrepancies between observation and prediction are associated with the inorganic soil ¹⁵N pool. We conclude that models in which fluxes are described using simple equations that can be augmented to include additional complexity, are an ideal starting point from which the relationship between GOF and model generalisability can be assessed.     

Serological survey for rabies antibodies in raptors from California

Fifty-three newly captive birds of prey were tested serologically for neutralizing antibodies against rabies virus, using a fluorescent focus inhibition test. No significant antibody titers were detected with this sensitive and specific technique in any of these birds. This study supports the contention that free-ranging birds of prey are of limited importance in the epidemiology of rabies.