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Coding 3 -exon is increasing 6 9 ; . The efficiency of polyadenylation at various sites and or the stability of mRNA polyadenylated at various sites have been shown to change with the metabolic state of a cell 6 ; or with stages of cell differentiation 8 ; . The molecular basis of these observations, however, is currently not well understood. Furthermore, the potential effect of drugs and environmental chemicals on alternative polyadenylation has not yet been explored in detail. The following communication details a more in-depth continuation of our previous investigations into pentobarbital PB ; 1mediated mRNA stability. Our results have directed us to focus attention on this region of the gstD21 mRNA molecule, the 3 -UTR as a possible critical site on mRNA stability, and alternative polyadenylation. The glutathione S-transferases GSTs, EC 2.5.1.18 ; are important Phase II detoxification enzymes 10 ; , and all are products of a gene superfamily 11 ; . The 10-member Drosophila gstD gene family 1214 ; includes gstD1 and gstD21 genes, which we have been using as reporters for investigating druginduced changes in mRNA stability 1516 ; . GST D1, the product of gstD1, is a DDT dehydrochlorinase, which converts DDT to DDE; its homolog gstD21 encodes a glutathione conjugatebinding protein with very low enzyme activities 17 ; . We have determined that PB, a general anesthetic, induces the expression of Drosophila gstD genes. This drug has been observed to induce gstD21 mRNA 10-fold, but most of this increase was thought to have come from changes in mRNA stability 15 ; . In this communication, we report the mapping of two gstD21 mRNAs L and S ; , which differ in polyadenylation sites. Both forms are equally inducible by PB, with the ratio L S ; holding at relatively constant proportions in the wild type flies. We have reconstituted the PB-mediated gstD21 mRNA stabilization in a heterologous context and designed experiments that focus on the importance of the native 3 -untranslated region 3 -UTR ; in determining gstD21 mRNA stability. We then used the induction and decay of this labile transgenic mRNA sequence to probe the effect of pentobarbital on alternative polyadenylation of the native gstD21 mRNAs. Our results show that a drug such as pentobarbital may regulate alternative polyadenylation. Moreover, this PB regulation appears to involve an important role for the level of the 3 -UTR sequence as a critical element recognized by the polyadenylation machinery Procedure Code Description INJECTION, PENICILLIN G BENZATHINE, UP TO 1, 200, 000 UNITS INJECTION, PENICILLIN G BENZATHINE, UP TO 2, 400, 000 UNITS INJECTION, PENICILLIN G BENZATHINE, UP TO 600, 000 UNITS INJECTION, PENICILLIN G POTASSIUM, UP TO 600, 000 UNITS INJECTION, PENICILLIN G PROCAINE, AQUEOUS, UP TO 600, 000 UNITS INJECTION, PENTASTARCH, 10% SOLUTION, 100 ML INJECTION, PENTAZOCINE, 30 MG INJECTION, PENTOBARBITAL SODIUM, PER 50 MG INJECTION, PHENOBARBITAL SODIUM, UP TO 120 MG INJECTION, PHENTOLAMINE MESYLATE, UP TO 5 MG INJECTION, PHENYLEPHRINE HCL, UP TO 1 ML INJECTION, PHENYTOIN SODIUM, PER 50 MG INJECTION, PHYTONADIONE VITAMIN K ; , PER 1 MG INJECTION, PIPERACILLIN SODIUM, 500 MG INJECTION, PIPERACILLIN SODIUM TAZOBACTAM SODIUM, 1 GRAM 0.125 GRAMS 1.125 GRAMS ; INJECTION, POTASSIUM CHLORIDE, PER 2 MEQ INJECTION, PRALIDOXIME CHLORIDE, UP TO 1 GM INJECTION, PREDNISOLONE ACETATE, UP TO 1 ML INJECTION, PROCAINAMIDE HCL, UP TO 1 GM INJECTION, PROCHLORPERAZINE, UP TO 10 MG INJECTION, PROGESTERONE, PER 50 MG INJECTION, PROMAZINE HCL, UP TO 25 MG INJECTION, PROMETHAZINE HCL, UP TO 50 MG INJECTION, PROPRANOLOL HCL, UP TO 1 MG INJECTION, PROTAMINE SULFATE, PER 10 MG INJECTION, PROTIRELIN, PER 250 MCG INJECTION, PYRIDOXINE HCL, 100 MG INJECTION, RANITIDINE HYDROCHLORIDE, 25 MG INJECTION, RASBURICASE, 0.5 MG INJECTION, RETEPLASE, 18.1 MG INJECTION, RHO D IMMUNE GLOBULIN, HUMAN, FULL DOSE, 300 MCG INJECTION, RHO D IMMUNE GLOBULIN, HUMAN, MINIDOSE, 50 MCG INJECTION, RHO D IMMUNE GLOBULIN, INTRAVENOUS, HUMAN, SOLVENT DETERGENT, 100 IU INJECTION, RISPERIDONE, LONG ACTING, 0.5 MG INJECTION, ROPIVACAINE HYDROCHLORIDE, 1 MG INJECTION, SARGRAMOSTIM GM-CSF ; , 50 MCG INJECTION, SINCALIDE, 5 MICROGRAMS INJECTION, SODIUM CHLORIDE, 0.9%, PER 2 ML INJECTION, SODIUM FERRIC GLUCONATE COMPLEX IN SUCROSE INJECTION, 12.5 MG.

Rized in table 2, there are differences among the barbiturates and dicarbamates in their relative potencies as potentiators of GABA P ; and direct GABAA receptor agonists A ; . Whereas the sedative-hypnotic compounds pentobarbital and meprobamate have low A P ratios 3 ; , the less sedative drug phenobarbital has a higher ratio 6 ; . Moreover, felbamate, which is not generally sedating but is frequently associated with insomnia Leppik and Wolff, 1995 ; , had no measurable agonist activity A P ratio 30 ; . We therefore propose that GABAA receptor positive modulators that produce direct receptor activation at concentrations closer to the concentrations producing GABA modulation low A P ratio as in table 2 ; have greater sedative activity. This implies that direct GABAA receptor activation is important to the powerful sedative-anesthetic effects of barbiturates such as pentobarbital and the dicarbamate meprobamate. In fact, at serum meprobamate concentrations associated with coma in man [120 mg l 549 M Bailey, 1981], the drug would be expected to have such a direct agonist activity. The deactivation time constants of currents activated by felbamate and meprobamate in the presence of GABA were substantially longer than the deactivation time constants of currents activated by GABA or the dicarbamates alone. Previously, we reported that phenobarbital markedly prolongs the decay of GABAA receptor currents, implying that pentobarbital and GABA can mutually stabilize binding to their respective recognition sites Rho et al., 1996 ; . In fact, the prolongation of the GABAA receptor current deactivation rate by felbamate and meprobamate was similar to that previously observed for pentobarbital time constant, 480 ms; Rho et al., 1996 ; . Thus, felbamate and meprobamate appear to share with pentobarbital the property of mutual stabilization of GABA binding. Our data from single-channel recordings provide additional evidence for the barbiturate-like nature of the GABAA receptor potentiation produced by the dicarbamates. Both felbamate and meprobamate prolonged the mean burst duration of control GABA currents in a similar fashion to barbiturates Macdonald et al., 1989b; Twyman et al., 1989a; Macdonald and Twyman, 1992; Rho et al., 1996 ; . In addition, at the high concentrations 3 mM ; used in our experiments, both felbamate and meprobamate induced flickering of the GABAA receptor currents, compatible with the rapid channel block we propose as a mechanism to explain the off-effect in the whole-cell recording experiments. A similar flickery block was observed previously with high concentrations of phenobarbital and pentobarbital Rho et al., 1996 ; . If entry of the.

1Journal Paper no. 1185, Purdue Agricultural Experiment Station. The experi mental data in this paper are taken from a thesis submitted by B. Bressani in partial fulfillment of the requirements for the Ph.D. degree in Biochemistry. A preliminary report appeared in Federation Proc., 15: 544, 1956. 'Rockefeller Fellow in Biochemistry. Present address: Institute of Nutrition of Central America and Panama INCAP ; , 481 Guatemala City, C. A. Adult SpragueDawley rats 60 days or older ; were anesthetized with sodium pentobarbital 55 mg kg ; . The motor branch of the pudendal nerve, which contains SNB axons, was exposed as it passes Cowper's gland just before it enters the BC LA muscle complex. Activity Block. The pudendal nerve was fitted with a cylindrical Silastic cuff 45 mm, 1.6 mm i.d., 3.2 mm o.d. ; attached via Silastic tubing 0.64 mm i.d., 1.2 mm o.d. ; to a subcutaneously implanted mini-osmotic pump total volume 100 l, rate 12 l per day; Alza ; . In experimental animals n 5 ; , the pumps contained 500 mg ml TTX Sigma ; in Hanks' balanced salt solution containing 200 units of penicillin and 200 g of streptomycin. Control animals n 5 ; received pumps filled with vehicle only. Five days later, animals were anesthetized and injected bilaterally with wheat-germ agglutinin conjugated to horseradish peroxidase WGA-HRP; Sigma ; into the BC LA muscle complex 2.5% in saline, 8 l per side ; . The ability of the motoneurons to transport WGA-HRP retrogradely to the cell body allowed an assessment of physical damage caused by the placement of the Silastic cuff. Approximately 20 h after WGA-HRP injections, the activity block was tested in each subject by visual evaluation of muscle response to electrical stimulation of the cuffed pudendal nerve. The animals were then given an overdose of sodium pentobarbital and perfused with heparinized saline followed by 5% phosphate-buffered acrolein. After perfusion, the lower lumbar spinal cord was removed, postfixed for 2 h, immersed in 20% sucrose overnight, and sectioned horizontally 40 m ; on freezing microtome. Alternate sections were stained immunocytochemically for AR with a 3, -diaminobenzidine DAB ; reaction see below ; , mounted on subbed slides, and counterstained with cresyl violet. The other sections were reacted with DAB to label the retrogradely transported WGA-HRP, then counterstained immunocytochemically for AR with nickelintensified DAB see below ; , and mounted on subbed slides. Blockade of Axoplasmic Transport. The pudendal nerve was exposed unilaterally and wrapped for 15 min with a 2-mg piece of cotton wool soaked in either 50 l of 300 M VBL Sigma ; in 0.9% saline n 6 ; or saline alone n 6 ; . The cotton-wrapped nerve was isolated from surrounding tissues by a piece of Parafilm. This treatment significantly disrupts axonal flow for at least 45 days with little morphological damage to the axon 44, 50 ; . Five days later, animals were anesthetized and injected bilaterally with WGA-HRP into the BC LA muscle complex to check for the effectiveness of VBL and peppermint.

A. fumigatus strain 13073 American Type Culture Collection, Manassas, VA ; was used in all studies, as this strain has previously been shown to induce invasive aspergillosis in immunocompromised mice 49 ; . The organism was grown on Sabouraud dextrose agar plates Becton Dickinson, Cockeysville, MD ; for 710 days at 37C. The surface of each plate was then washed with 100 ml of sterile 0.1% Tween 80 Sigma, St. Louis, MO ; in normal saline. The resulting suspension of conidia was filtered through sterile gauze to remove clumps and hyphal debris, and then washed once and resuspended in 4 ml 0.1% Tween 80. The concentration of Aspergillus conidia in the suspension was determined using a particle counter Z2 particle analyzer, Coulter, Hialeah, FL ; . The suspension was then diluted to the desired concentration, and the concentration was again measured before administration. In preliminary experiments the number of particles determined by the particle counter was in close agreement with the number of viable CFU found by serial dilution and plating of the suspension. On the day of inoculation, each animal was anesthetized with 1.8 2 mg of pentobarbital i.p. Using standard aseptic technique, the trachea was exposed, and a 30- l inoculum A. fumigatus suspension or 0.1% Tween 80 vehicle ; was administered via a sterile 26-gauge needle. The skin incision was closed with surgical staples. Animals were challenged with inocula ranging.

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