In Vivo Microdialysis Studies on the Effects of Decortication and Excitotoxic Lesions on Kainic Acid-Induced Calcium Fluxes, and Endogenous Amino Acid Release, in the Rat Striatum

The in vivo effects of kainate (1 mM) on fluxes of 45Ca2+, and endogenous amino acids, were examined in the rat striatum using the brain microdialysis technique. Kainate evoked a rapid decrease in dialysate 45Ca2+, and an increase in the concentration of amino acids in dialysates in Ca2+-free dialysates. Taurine was elevated six- to 10-fold, glutamate two- to threefold, and aspartate 1.5- to twofold. There was also a delayed increase in phosphoethanolamine, whereas nonneuroactive amino acids were increased only slightly. The kainic acid-evoked reduction in dialysate 45Ca2+ activity was attenuated in striata lesioned previously with kainate, suggesting the involvement of intrinsic striatal neurons in this response. The increase in taurine concentration induced by kainate was slightly smaller under these conditions. Decortication did not affect the kainate-evoked alterations in either dialysate 45Ca2+ or amino acids. These data suggest that kainate does not release acidic amino acids from their transmitter pools located in corticostriatal terminals.     

A Possible Interrelationship Between Extracellular Taurine and Phosphoethanolamine in the Hippocampus

The effect of guanidinoethane sulfonic acid (GES), an inhibitor of taurine uptake, was examined with respect to endogenous amino acids in the hippocampus of the freely moving rabbit. GES increased the extracellular levels of both taurine and phosphoethanolamine (PEA), other amino acids being unaffected. However, long-term oral administration of GES selectively reduced endogenous taurine levels. The effect of GES on PEA appeared to be a consequence of the elevated extracellular taurine as exogenously administered taurine per se increased PEA levels in the extracellular space. The findings are discussed in conjunction with the proposed membrane-stabilizing effects of taurine.     

High-Purity Preparation of HSV-2 Vaccine Candidate ACAM529 Is Immunogenic and Efficacious In Vivo

Genital herpes is a sexually transmitted infection (STI) caused by herpes simplex virus 2 (HSV-2) and to a lesser extent herpes simplex virus 1 (HSV-1). Infection by HSV-2 is life-long and is associated with significant cost to healthcare systems and social stigma despite the highly prevalent nature of the disease. For instance, the proportion of HSV-2 seropositive to seronegative adults is approximately 1 in 5 in the US and greater than 4 in 5 in some areas of sub-Saharan Africa. The replication-defective vaccine strain virus dl5-29 was re-derived using cells appropriate for GMP manufacturing and renamed ACAM529. Immunization with dl5-29 was previously reported to be protective both in mice and in guinea pigs, however these studies were performed with vaccine that was purified using methods that cannot be scaled for manufacturing of clinical material. Here we describe methods which serve as a major step towards preparation of ACAM529 which may be suitable for testing in humans. ACAM529 can be harvested from infected cell culture of the trans-complementing cell line AV529 clone 19 (AV529-19) without mechanical cell disruption. ACAM529 may then be purified with respect to host cell DNA and proteins by a novel purification scheme, which includes a combination of endonuclease treatment, depth filtration, anion-exchange chromatography and ultrafiltration/diafiltration (UF/DF). The resultant virus retains infectivity and is ∼ 200-fold more pure with respect to host cell DNA and proteins than is ACAM529 purified by ultracentrifugation. Additionally, we describe a side-by-side comparison of chromatography-purified ACAM529 with sucrose cushion-purified ACAM529, which shows that both preparations are equally immunogenic and protective when tested in vivo.     

NEURONAL AND GLIAL SYSTEMS FOR γ-AMINOBUTYRIC ACID METABOLISM

—Bulk prepared neuronal perikarya, nerve endings and glial cells have been used to study amino acid concentrations and GABA metabolism in vitro. All amino acids were more concentrated in synaptosomes and glial cells than in neuronal perikarya. Cell specificity was found with respect to the relative distribution of some amino acids. Glutamate decarboxylase activity was considerably higher in synaptosomes than in glial cells. The inhibitory effect of amino-oxyacetic acid on glutamate decarboxylase activity differed between synaptosomes and glial cells. γ-Aminobutyric acid-α-ketoglutarate transaminase had the highest activity in the glial cell fraction; the inhibition of amino-oxyacetic acid differed between glial and neuronal material. The metabolism of exogenous GABA just accumulated by a cell showed similar time characteristics in neuronal and glial material.     

PROTEIN TURNOVER IN CELL-ENRICHED FRACTIONS FROM RABBIT BRAIN1

Abstract— A modification of the available methods was used for preparation of nerve and glia cell-enriched fractions from rabbit brain and spinal cord. The rate of incorporation of tritiated leucine and the turnover rates of protein during 10 days was studied in the bulkprepared cell fractions. The rate of incorporation into the nerve cell fraction was approximately three times greater than in the glia fraction. The nerve cells had one rapid and one slow phase in decline of radioactivity, while the glial cells were characterized by a more uniform decline. The soluble radioactivity was followed in whole tissue of brain and spinal cord and certain differences between the two were observed.     

Use of Ecotilling as an efficient SNP discovery tool to survey genetic variation in wild populations of Populus trichocarpa

Abstract Ecotilling was used as a simple nucleotide polymorphism (SNP) discovery tool to examine DNA variation in natural populations of the western black cottonwood, Populus trichocarpa, and was found to be more efficient than sequencing for large-scale studies of genetic variation in this tree. A publicly available, live reference collection of P. trichocarpa from the University of British Columbia Botanical Garden was used in this study to survey variation in nine different genes among individuals from 41 different populations. A large amount of genetic variation was detected, but the level of variation appears to be less than in the related species, Populus tremula, based on reported statistics for that tree. Genes examined varied considerably in their level of variation, from PoptrTB1 which had a single SNP, to PoptrLFY which had more than 23 in the 1000-bp region examined. Overall nucleotide diversity, measured as (Total), was relatively low at 0.00184. Linkage disequilibrium, on the other hand, was higher than reported for some woody plant species, with mean r2 equal to 0.34. This study reveals the potential of Ecotilling as a rapid genotype discovery method to explore and utilize the large pool of genetic variation in tree species.     

Mechanisms of ATP-mediated vasodilation in humans: modest role for nitric oxide and vasodilating prostaglandins

ATP is an endothelium-dependent vasodilator, and findings regarding the underlying signaling mechanisms are equivocal. We sought to determine the independent and interactive roles of nitric oxide (NO) and vasodilating prostaglandins (PGs) in ATP-mediated vasodilation in young, healthy humans and determine whether any potential role was dependent on ATP dose or the timing of inhibition. In protocol 1 (n = 18), a dose-response curve to intrabrachial infusion of ATP was performed before and after both single and combined inhibition of NO synthase [N(G)-monomethyl-L-arginine (L-NMMA)] and cyclooxygenase (ketorolac). Forearm blood flow (FBF) was measured via venous occlusion plethysmography and forearm vascular conductance (FVC) was calculated. In this protocol, neither individual nor combined NO/PG inhibition had any effect on the vasodilatory response (P = 0.22-0.99). In protocol 2 (n = 16), we determined whether any possible contribution of both NO and PGs to ATP vasodilation was greater at low vs. high doses of ATP and whether inhibition during steady-state infusion of the respective dose of ATP impacted the dilation. FBF in this protocol was measured via Doppler ultrasound. In protocol 2, infusion of low (n = 8)- and high-dose (n = 8) ATP for 5 min evoked a significant increase in FVC above baseline (low = 198 ± 24%; high = 706 ± 79%). Infusion of L-NMMA and ketorolac together reduced steady-state FVC during both low- and high-dose ATP (P < 0.05), and in a subsequent trial with continuous NO/PG blockade, the vasodilator response from baseline to 5 min of steady-state infusion was similarly reduced for both low (ΔFVC = -31 ± 11%)- and high-dose ATP (ΔFVC -25 ± 11%; P = 0.70 low vs. high dose). Collectively, our findings indicate a potential modest role for NO and PGs in the vasodilatory response to exogenous ATP in the human forearm that does not appear to be dose or timing dependent; however, this is dependent on the method for assessing forearm vascular responses. Importantly, the majority of ATP-mediated vasodilation is independent of these putative endothelium-dependent pathways in humans.     

Automated measurements of antilisterial activities of lactate and diacetate in ready-to-eat meat

Standard procedures to enumerate Listeria organisms rely on plating food samples on selective agar media. The procedures are labor-intensive, and because of the limited sensitivity, pre-enrichment step is required for the detection of low numbers of the pathogen. In the present study, an automated rapid optic procedure and the standard procedure were used to determine the behavior of the pathogen in ready-to-eat (RTE) meat and to test the effect of antilisterial agents. Listeria monocytogenes strain Scott A or a six-strain mixture of Listeria was studied using lactate (2.5%), diacetate (0.2%) and their combination in beef bologna and in sterile beef emulsion. Samples stored for up to 60 days at 5 and 10 degrees C were tested at time intervals during storage. Using the plate count method, each of the salts caused a delay in growth of the pathogen, and the salt combination was most effective causing listeriostatic effects and decline in growth of the pathogen at 5 degrees C. High negative correlation (r), ranging from 0.92 to 0.99, was obtained between the detection time (DT) recorded by the optic procedure (BioSys instrument) and cell numbers determined by the plate count procedure. The rapid (< 24 h) optic procedure was reliable in assessing the efficacy of antimicrobials and in rapid detection of low levels of listeriae that are undetectable by direct plating procedure.