Cyclosporin A (CsA) is known to possess antiviral activity against several viruses in vitro, but the effect of CsA on BK polyoma virus (BKV) replication has not been examined. We investigated the impact of CsA on primary, chronic, and high-level BKV infection using a cell system of kidney cell origin (Vero E6 cells). During the first 2 h post infection, cells treated with CsA up to 3200 microg/L showed a near-identical BK viral load to untreated cells, with only a very minor reduction in the CsA-treated cells observed at 4 h. In chronic culture, CsA completely suppressed the primary BKV infection peak in a non-dose-dependent manner within the dose range of 200-12,800 microg/L (P<0.05). BKV reactivation was also inhibited in the presence of CsA at doses of 200-3200 microg/L: the mean number of BKV DNA copies/mL remained stable or even decreased slightly compared with a 7-log increase in the non-CsA group (P<0.01). CsA did not influence BKV DNA copies/mL in Vero E6 cells with high-level infection (>10(9) copies/mL). Cellular protein measurements indicated that the antiviral effect of CsA was not a result of cytotoxicity. These findings from a relevant in vitro kidney cell system indicate that CsA suppresses the primary BKV infection peak and inhibits escape to BKV reactivation; these effects are dose independent and not related to cytotoxicity. The intracellular antiviral mode of action of CsA against BKV does not appear to be via inhibition of viral cell entry pathways.

Pamidronate is a bisphosphonate that is effective in treating bone disease including osteopenia and osteoporosis in adults. A sensitive and reliable method for the analysis of pamidronate in whole blood and urine is key to the development of this drug for use in children. A previously described method for pamidronate analysis serum and urine did not consistently detect the drug at satisfactory levels in whole blood. The procedure involves co-precipitation of the bisphosphonates with calcium phosphate, pre-column derivitization with fluorescamine, HPLC utilizing a Nucleosil C(18) column, and fluorescence detection with excitation at 395 nm and emission at 480 nm. Changes to the original protocol included the use of a new internal standard (alendronate), the optimization of the concentration of ethylenediaminetetraacetic acid (EDTA) for dissolving the precipitate, and the elimination of the acidification step prior to deproteinization. The optimum EDTA concentration, which had a significant effect on the labeling capability of fluorescamine, was determined to be 20 mm.A good separation between pamidronate and alendronate was achieved using a heated (40 degrees C ) Nucleosil C(18), 10 micro m particle size column. The mobile phase was an aqueous solution of 1 mm Na(2)EDTA-methanol (97:3, v/v) adjusted to pH 6.5 using a fl ow-rate of 1 mL/min. Fluorescence detection was set at 395 nm for excitation and at 480 nm for emission. The limit of quantitation for pamidronate was 0.5 micro g/mL in whole blood and 0.1 micro g/mL in urine. The method was applied to both whole blood and urine samples from pediatric patients.

No abstract

To characterize the effect of glutamine on the release of glutamate, aspartate, and gamma-aminobutyric acid (GABA), rat hippocampal slices were superfused with different concentrations of glutamine or Ca2+. Amino acids released and retained were analyzed by HPLC. Glutamine (0.5 mmol/L) increased more than threefold the release of glutamate evoked by 50 mmol/L K+ in the presence of 2.6 mmol/L Ca2+ without a corresponding increase in glutamate content, while the release of aspartate was increased less and that of GABA not at all by glutamine. The evoked release of all three amino acids, including the enhanced release of glutamate in the presence of glutamine, was strongly dependent on Ca2+ concentrations between 0.1 and 2.6 mmol/L. The potentiation of glutamate release by glutamine reached a plateau at 0.25 mmol/L glutamine. Intermittent electrical field stimulation increased the release of only glutamate and this release was nearly doubled by glutamine. The increased release was Ca2+ dependent and tetrodotoxin (TTX) sensitive. Results suggest that extracellular glutamine promotes primarily the formation of releasable glutamate and this enhancement is dependent on extracellular Ca2+.

It is known that low glucose concentrations increase the aspartate and decrease the glutamate content of brain tissue both in vivo and in vitro. To see whether these changes occur in the transmitter compartment or not, the release of aspartate and glutamate evoked by electrical-field stimulation or by high K+ was followed in slices of rat hippocampus superfused with 5 or 0.2 mM glucose. Superfusion with 0.2 mM glucose increased the evoked release of aspartate about ten times and that of glutamate about threefold. This shift in the ratio of aspartate to glutamate released was accompanied by a similar increase in the relative amount of aspartate contained in the slices. The high evoked release of aspartate and glutamate was well maintained, provided 0.5 mM glutamine was added to the medium. Changing the concentration of glucose after the first period of stimulation rapidly altered the relative amounts of aspartate and glutamate released but not the enhanced release of glutamate. The large evoked release of both aspartate and glutamate in 0.2 mM glucose was almost entirely Ca2+-dependent. The relative amounts of aspartate and glutamate released by 50 mM K+ also changed when the glucose concentration was reduced. Results suggest two effects of low glucose concentrations: an increase in the overflow of synaptically released glutamate due to a decreased uptake and an increase in the proportion of aspartate to glutamate formed and released from the transmitter pool. These observations are consistent with the interpretation that these two transmitters can be released in different proportions from the same terminals.