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.      

In situ hybridization at the electron microscope level: hybrid detection by autoradiography and colloidal gold

In situ hybridization has become a standard method for localizing DNA or RNA sequences in cytological preparations. We developed two methods to extend this technique to the transmission electron microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope level using mouse satellite DNA hybridization to whole mount metaphase chromosomes as the test system. The first method devised is a direct extension of standard light microscope in situ hybridization. Radioactively labeled complementary RNA (cRNA) is hybridized to metaphase chromosomes deposited on electron microscope grids and fixed in 70 percent ethanol vapor; hybridixation site are detected by autoradiography. Specific and intense labeling of chromosomal centromeric regions is observed even after relatively short exposure times. Inerphase nuclei present in some of the metaphase chromosome preparations also show defined paatterms of satellite DNA labeling which suggests that satellite-containing regions are associate with each other during interphase. The sensitivity of this method is estimated to at least as good as that at the light microscope level while the resolution is improved at least threefold. The second method, which circumvents the use of autoradiogrphic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction is improved at least threefold. The second method, which circumvents the use of autoradiographic detection, uses biotin-labeled polynucleotide probes. After hybridization of these probes, either DNA or RNA, to fixed chromosomes on grids, hybrids are detected via reaction with an antibody against biotin and secondary antibody adsorbed to the surface of over centromeric heterochromatin and along the associated peripheral fibers. Labeling is on average ten times that of background binding. This method is rapid and possesses the potential to allow precise ultrastructual localization of DNA sequences in chromosomes and chromatin.     

Molecular evolution in the gnd locus of Salmonella enterica

The gnd gene, the structural gene for 6-phosphogluconate dehydrogenase, was sequenced and analyzed in 34 isolates from different serovars of the seven subspecies of Salmonella enterica to provide comparative information on the evolution in this gene, which has been studied extensively in Escherichia coli. The gene tree obtained by the neighbor- joining method in general gave separate branches for each subspecies, with the few exceptions readily explained by recombination. There is evidence of recombination involving transfer of long (more than 400 bp) and short (30-150 bp) segments of DNA. Four of the six long-segment transfers detected are at the 5' end of the gene, and in all four cases a variant of the chi sequence is located close to the recombination junction and appears to have mediated the recombination events. We suggest that in these four cases and in a fifth case with intersubspecies transfer of the whole gnd gene, the adjacent rfb (O antigen) locus may have been transferred in the same event. The estimates of the number of synonymous substitutions per synonymous site, KS, and the number of nonsynonymous substitutions per nonsynonymous site, KA, within the E. coli and S. enterica gnd genes, and also between the two species show an interesting distribution, with KS being lower toward the ends of the gene and KA in particular being lower in the first than in the second domain. In S. enterica, synonymous sites also seem to be subjected to negative selection. The ratio of KA to KS was higher within S. enterica and E. coli than between them, which may indicate that intraspecies variation is essentially between clones and that mildly deleterious mutations can be fixed within clones, which would thus raise KA within species.      

Localization of acetylcholine receptors and synaptic ultrastructure at nerve-muscle contacts in culture: dependence on nerve type

In cultures of xenopus myotomal muscle cells and spinal cord (SC) some of the nerve-muscle contacts exhibit a high density of acetylcholine receptors (AchRs [Anderson et al., 1977, J. Physiol. (Lond.). 268:731- 756,757-773]) and synaptic ultrastructure (Weldon and Cohen, 1979, J. Neurocytol. 8:239-259). We have examined whether similarly specialized contacts are established when the muscle cells are cultured with explants of xenopus dorsal root ganglia (DRG) or sympathetic ganglia (SG). The outgrowth from the ganglionic explants contained neuronal and non- neuronal cell processes. Although both types of processes approached within 100 A of muscle cells, synaptic ultrastructure was rarely observed at these contacts. Because patches of postsynaptic ultrastructure also develop on noncontacted muscle cells, the very few examples of contacts with such specializations probably occurred by chance. AChRs were stained with fluroscent α-bungarotoxin. More than 70 percent of the SC-contacted muscle cells exhibited a high receptor density along the path of contact. The corresponding values for DRG- and SG- contacted muscle cells were 10 and 6 percent. Similar values were obtained when the ganlionic and SC explants were cultured together in the same chamber. The few examples of high receptor density at ganglionic-muscle contacts resembled the characteristic receptor patches of noncontacted muscle cells rather than the narrow bands of high receptor density seen at SC-muscle contacts. In addition, more than 90 percent of these ganglionic- contacted muscle cells had receptor patches elsewhere, compared to less than 40 percent for the SC-contacted muscle cells. These findings indicate that the SC neurites possess a specific property which is important for the establishment of synaptically specialized contacts with muscle and that this property is lacking in the DRG and SG neurites.     

Biofilter efficiency of <i>Eichhornia crassipes</i> in wastewater treatment of fish farming in Amazonia

Fish is a very important part of the human diet in Amazonia. Near the growing cities, fish populations and individual size have decreased over the past decades. Alternatives to traditional and industrial fishing arise, including fish farming. Strategies to minimize the impact of fish farms on the environment are needed to have a regular and healthy fish supply. This is to avoid a reduction of biodiversity, a depletion of natural resources, and/or the induction of significant changes in the structure and functioning of adjacent ecosystems. Very little research has been performed on management of effluents as to maintain the quality of water resources. The present study aimed at testing the efficiency of the Amazonian aquatic macrophyte Eichhornia crassipes as a biofilter for the treatment of effluents from fish farming. In three filtering treatments (50%, 75% and 100% plant cover) and a control (0%), physical and chemical properties of the water were measured and analyzed in a nursery with fish after passing the biofilter system, with a hydraulic retention time of 24 hours. The analyzed variables showed no significant differences (p>0.05) among the treatments with 50-100% cover, indicating that 50% cover would be enough for a good efficiency of the biofilter. All parameters were reduced after passage of the biofilter under the presence of E. crassipes: 73.7% for electrical conductivity, 15% for pH, 84.5% for turbidity, 86.8% for nitrite, 69% for total phosphorus, and 77.8% for orthophosphate. The concentrations of total nitrogen, nitrate and ammonium ions were not significantly changed (p>0.05). We conclude that E. crassipes is effective in improving the quality of effluents from fish farming, with less efficiency for nitrogen compounds. Our treatment system can be adopted by small and medium-sized farmers, aiming at a sustainable employment of the activity.