GLT-1, GLAST, and EAAC1 are high-affinity, Na(+)-dependent glutamate transporters identified in rat forebrain. The expression of these transporter subtypes was characterized in three preparations: undifferentiated rat cortical astrocyte cultures, astrocytes cocultured with cortical neurons, and astrocyte cultures differentiated with dibutyryl cyclic AMP (dBcAMP). The undifferentiated astrocyte monocultures expressed only the GLAST subtype. Astrocytes cocultured with neurons developed a stellate morphology and expressed both GLAST and GLT-1; neurons expressed only the EAAC1 transporter, and rare microglia in these cultures expressed GLT-1. Treatment of astrocyte cultures with dBcAMP induced expression of GLT-1 and increased expression of GLAST. These effects of dBcAMP on transporter expression were qualitatively similar to those resulting from coculture with neurons, but immunocytochemistry showed the pattern of transporter expression to be more complex in the coculture preparations. Compared with astrocytes expressing only GLAST, the dBcAMP-treated cultures expressing both GLAST and GLT-1 showed an increase in glutamate uptake Vmax, but no change in the glutamate K(m) and no increased sensitivity to inhibition by dihydrokainate. Pyrrolidine-2,4-dicarboxylic acid and threo-beta-hydroxyaspartic acid caused relatively less inhibition of transport in cultures expressing both GLAST and GLT-1, suggesting a weaker effect at GLT-1 than at GLAST. These studies show that astrocyte expression of glutamate transporter subtypes is influenced by neurons, and that dBcAMP can partially mimic this influence. Manipulation of transporter expression in astrocyte cultures may permit identification of factors regulating the expression and function of GLAST and GLT-1 in their native cell type.

Both acidosis and oxidative stress contribute to ischemic brain injury. The present study examines interactions between acidosis and oxidative stress in murine cortical cultures. Acidosis (pH 6.2) was found to potentiate markedly neuronal death induced by H 2 O 2 exposure. To determine if this effect was mediated by decreased antioxidant capacity at low pH, the activities of several antioxidant enzymes were measured. Acidosis was found to reduce the activities of glutathione peroxidase and glutathione S-transferase by 50 -60% ( p Ͻ 0.001) and the activity of glutathione reductase by 20% ( p Ͻ 0.01) in lysates of the cortical cultures. Like acidosis, direct inhibition of glutathione peroxidase with mercaptosuccinate also potentiated H 2 O 2 toxicity. Because acidosis may accelerate hydroxyl radical production by the Fenton reaction, the effect of iron chelators was also examined. Both desferrioxamine and N,N,NЈ,NЈ-tetrakis(2-pyridylmethyl)ethylenediamine, two structurally different iron chelators, significantly reduced H 2 O 2 -induced neuronal death under both pH 7.2 and pH 6.2 conditions. These results suggest that the increased cell death produced by severe acidosis during cerebral ischemia may result in part from exacerbation of oxidative injury. This exacerbation may result from both impaired antioxidant enzyme functions and increased intracellular free iron levels.

A protocol for quantification of human immunodeficiency virus type 1 (HIV-1) proviral DNA with the TaqMan technology was developed and validated. The assay was specific for HIV-1, with an analytic sensitivity of 10 copies and a linear dynamic range of >6 logs. Viral RNA levels, when at a stable state, were highly correlated with proviral DNA levels in 80 specimens of 18 HIV-infected children.The use of highly active antiretroviral therapy (HAART) in human immunodeficiency virus (HIV)-infected patients can often significantly reduce the levels of viral RNA to undetectable levels in plasma in both adults and children (13)(14)(15)(16)(17). However, it is less clear how changes in proviral DNA levels respond to HAART in infected patients or what relative changes in proviral DNA levels precede changes in viral RNA levels after HAART induction. One of the current challenges is detection of low levels of proviral DNA in latently infected CD4 ϩ lymphocytes and other reservoirs, which can replenish and revive viral infection upon activation (3-5). Thus, a highly reproducible and accurate assay for quantification of proviral DNA would enable more in-depth evaluations of the efficacies of antiviral therapies.Several assays for the quantification of HIV type 1 (HIV-1) proviral DNA have previously been reported, and all of these were based on the principle of conventional PCR (1, 2, 6, 10, 11). The potential limitation associated with the traditional quantitative PCR is that the DNA copy numbers are calculated on the basis of the quantities of the final amplified gene products. Since DNA is amplified exponentially during PCR, a small variation in amplification efficiency early in the thermocycling process could potentially lead to large variations in the final quantities of amplified products.The real-time PCR, which is also known as the TaqMan or the 5Ј exonuclease assay, quantifies PCR products cycle by cycle (in real time) as they accumulate (7-9). There are several advantages of using real-time PCR. (i) It does not rely on the final product of PCR amplification for quantification. DNA copy numbers are determined on the basis of the threshold cycle number (C T ), which is directly proportional to the initial copy number. (ii) It is relatively specific. If the probe binds nonspecifically to some sequences other than the target sequence, it will not be cleaved or detected as part of the amplification. (iii) It allows a wide dynamic range of detection since the measured DNA copy number is directly proportional to the initial copy number. (iv) All real-time PCR tests are performed in 96-well and closed-tube formats, allowing highthroughput testing and a greatly reduced chance of cross-contamination.In this report, a new protocol for quantification of HIV-1 proviral DNA with the TaqMan technology is described and validated. By using this method, HIV-1 proviral DNA levels were compared in parallel with the HIV-1 RNA levels at a stable state during HAART.(This study was part of a thesis by Johann Miller in partial fulfillment of...