Evidence for a sialic acid salvaging pathway in lepidopteran insect cells

We previously described a transgenic insect cell line, Sfbeta4GalT/ST6, that expresses mammalian beta-1,4-galactosyltransferase and alpha2,6-sialyltransferase genes and produces glycoproteins with terminally sialylated N-glycans. The ability of these cells to produce sialylated N-glycans was surprising because insect cells contain only small amounts of sialic acid and no detectable CMP-sialic acid. Thus, it was of interest to investigate potential sources of sialic acids for sialoglycoprotein synthesis by these cells. We found that Sfbeta4GalT/ST6 cells can produce sialylated N-glycans when cultured in the presence but not in the absence of fetal bovine serum. The serum component(s) supporting N-glycan sialylation by Sfbeta4GalT/ST6 cells is relatively large-it was not removed by dialysis in a 50,000-molecular-weight cutoff membrane. Serum-free media supplemented with purified fetuin but not asialofetuin supported N-glycan sialylation by Sfbeta4GalT/ST6 cells. The terminally sialylated N-glycans isolated from fetuin also supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells. Finally, serum-free medium supplemented with N-acetylneuraminic acid or N-acetylmannosamine supported glycoprotein sialylation by Sfbeta4GalT/ST6 cells but to a much lower degree than serum or fetuin. These results provide the first evidence of a sialic acid salvaging pathway in insect cells, which begins to explain how Sfbeta4GalT/ST6 and other transgenic insect cell lines can sialylate recombinant glycoproteins in the absence of a more obvious source of CMP-sialic acid.     

Ribozymes as tools for therapeutic target validation in arthritis

In this paper we describe a method for validating therapeutic gene targets in arthritic disease. Ribozymes are catalytic oligonucleotides capable of highly sequence-specific cleavage of RNA. We designed ribozymes that cleave the mRNA encoding stromelysin, a matrix metalloproteinase implicated in cartilage catabolism. Ribozymes were initially screened in cultured fibroblasts to identify sites in the mRNA that were accessible for binding and cleavage. Accessible sites for ribozyme binding were found in various regions of the mRNA, including the 5' untranslated region, the coding region, and the 3' untranslated region. Several ribozymes that mediated sequence-specific and dose-dependent inhibition of stromelysin expression were characterized. Site selection in cell culture was predictive of in vivo bioactivity. An assay for measuring cartilage catabolism in rabbit articular cartilage explants was developed. Ribozymes inhibited IL-1-stimulated stromelysin mRNA expression in articular cartilage explants, yet failed to inhibit proteoglycan degradation. This indicated that up-regulation of stromelysin was not essential for IL-1-induced cartilage catabolism. Broad applications of this approach in therapeutic target validation are discussed.     

Fast inactivation of voltage-dependent calcium channels. A hinged-lid mechanism?

We recently described domains II and III as important determinants of fast, voltage-dependent inactivation of R-type calcium channels (Spaetgens, R. L., and Zamponi, G. W. (1999) J. Biol. Chem. 274, 22428-22438). Here we examine in greater detail the structural determinants of inactivation using a series of chimeras comprising various regions of wild type alpha(1C) and alpha(1E) calcium channels. Substitution of the II S6 and/or III S6 segments of alpha(1E) into the alpha(1C) backbone resulted in rapid inactivation rates that closely approximated those of wild type alpha(1E) channels. However, neither individual or combined substitution of the II S6 and III S6 segments could account for the 60 mV more negative half-inactivation potential seen with wild type alpha(1E) channels, indicating that the S6 regions contribute only partially to the voltage dependence of inactivation. Interestingly, the converse replacement of alpha(1E) S6 segments of domains II, III, or II+III with those of alpha(1C) was insufficient to significantly slow inactivation rates. Only when the I-II linker region and the domain II and III S6 regions of alpha(1E) were concomitantly replaced with alpha(1C) sequence could inactivation be abolished. Conversely, introduction of the alpha(1E) domain I-II linker sequence into alpha(1C) conferred alpha(1E)-like inactivation rates, indicating that the domain I-II linker is a key contributor to calcium channel inactivation. Overall, our data are consistent with a mechanism in which inactivation of voltage-dependent calcium channels may occur via docking of the I-II linker region to a site comprising, at least in part, the domain II and III S6 segments.     

G protein modulation of N-type calcium channels is facilitated by physical interactions between syntaxin 1A and Gbetagamma

The direct modulation of N-type calcium channels by G protein betagamma subunits is considered a key factor in the regulation of neurotransmission. Some of the molecular determinants that govern the binding interaction of N-type channels and Gbetagamma have recently been identified (see, i.e., Zamponi, G. W., Bourinet, E., Nelson, D., Nargeot, J., and Snutch, T. P. (1997) Nature 385, 442-446); however, little is known about cellular mechanisms that modulate this interaction. Here we report that a protein of the presynaptic vesicle release complex, syntaxin 1A, mediates a crucial role in the tonic inhibition of N-type channels by Gbetagamma. When syntaxin 1A was coexpressed with (N-type) alpha(1B) + alpha(2)-delta + beta(1b) channels in tsA-201 cells, the channels underwent a 18 mV negative shift in half-inactivation potential, as well as a pronounced tonic G protein inhibition as assessed by its reversal by strong membrane depolarizations. This tonic inhibition was dramatically attenuated following incubation with botulinum toxin C, indicating that syntaxin 1A expression was indeed responsible for the enhanced G protein modulation. However, when G protein betagamma subunits were concomitantly coexpressed, the toxin became ineffective in removing G protein inhibition, suggesting that syntaxin 1A optimizes, rather than being required for G protein modulation of N-type channels. We also demonstrate that Gbetagamma physically binds to syntaxin 1A, and that syntaxin 1A can simultaneously interact with Gbetagamma and the synprint motif of the N-type channel II-III linker. Taken together, our experiments suggest a mechanism by which syntaxin 1A mediates a colocalization of G protein betagamma subunits and N-type calcium channels, thus resulting in more effective G protein coupling to, and regulation of, the channel. Thus, the interactions between syntaxin, G proteins, and N-type calcium channels are part of the structural specialization of the presynaptic terminal.     

Study of Toxin Production by Isolates of Stachybotrys chartarum and Memnoniella echinata Isolated during a Study of Pulmonary Hemosiderosis in Infants

A cluster of cases of pulmonary hemosiderosis among infants was reported in Cleveland, Ohio, during 1993 and 1994. These unusual cases appeared only in infants ranging in age from 1 to 8 months and were characterized by pulmonary hemorrhage, which caused the babies to cough up blood. A case-control study identified major home water damage (from plumbing leaks, roof leaks, or flooding) as a risk factor for development of pulmonary hemorrhage in these infants. Because of an interest in the possibility that trichothecene mycotoxins might be involved in this illness, a number of isolates of Stachybotrys chartarum were grown in the laboratory on rice, and extracts were prepared and analyzed both for cytotoxicity and for specific toxins. Two isolates of Memnoniella echinata, a fungus closely related to S. chartarum, were also included in these studies. S. chartarum isolates collected from the homes were shown to produce a number of highly toxic compounds, and the profiles of toxic compounds from M. echinata were similar; the most notable difference was the fact that the principal metabolites produced by M. echinata were griseofulvins.     

Denitrification and N2O emissions from a UK pasture soil following the early spring application of cattle slurry and mineral fertiliser

Total denitrification and nitrous oxide (N₂O) losses were measured from three contrasting dairy management systems representing good commercial practice (system 1), production maintained but with reduced N losses (system 2); and nitrate leaching less than 50 mg L^-1 but with reduced production (system 3). Measurements were made following mineral fertiliser application and from two plot experiments where four treatments were applied: control, NH₄NO₃ at 60 kg N ha^-1, cattle slurry applied to the surface (equivalent to 45 kg N ha^-1), and cattle slurry injected. Despite low soil temperatures (<6 °C) and low rainfall (<3 mm), total denitrification and N₂O losses peaked at 56 and 16 g N ha^-1 d^-1, respectively. Total denitrification losses decreased: system 1 system 2 > system 3, whereas N₂O losses decreased: system 2 > system 3 > system 1. Total denitrification losses tended to decrease with decreasing fertiliser application rate, whereas fertiliser application rate was not the sole determinant of the N₂O loss. The system 3 field was injected with cattle slurry for 2 yr, system 2 received some slurry by injection and system 1 received slurry to the surface. Thus, the amount, timing and method of previous cattle slurry application was important in determining the loss following subsequent fertiliser application. For the plot experiments, total denitrification and N₂O losses decreased in the order: slurry injected > mineral fertiliser > slurry applied to the surface > control for 5 days following application. However, 16 and 19 days after application, N₂O losses above the control were measured from plots that had received cattle slurry. It was inferred that the application of cattle slurry to the pasture soil stimulated greater N₂O production and increased losses over a longer time period compared with mineral fertiliser additions.     

Ultrastructure and chemistry of the cell wall of the moss Rhacocarpus purpurascens (Rhacocarpaceae): a puzzling architecture among plants

The architectural, compositional and functional characteristics of the cell walls of the leaves of the moss Rhacocarpus purpurascens (Brid.) Par. have been analysed by scanning and transmission electron microscopy, wall-extraction methods, nuclear magnetic resonance (NMR) spectroscopy, and water-retention experiments. Four-layered cell walls with a peculiar architecture which, so far, appears to be unique among plants were apparent. The architecture of the walls was not affected by sequential wall-extraction procedures. Subsequent analysis of the residual pre-extracted walls by classical spectro-photometrical methods revealed that the walls are composed of mainly lignin, hemicellulose and cellulose in a ratio of about 9:8:5, determining their integrity. This was supported by NMR spectroscopy. The resonance spectrum showed various characteristics typical of lignin; however, some specific peaks associated with lignin were missing. The walls exhibited no particular properties for external water conduction but seem to be adapted to rapid absorption of fog, dew, or rain. Received: 3 June 1997 / Accepted: 25 February 1998