A new reversed-phase high-pressure liquid chromatography assay procedure for dihydropteroate synthetase (DHPS) that involves the elution of the enzyme incubation solution with a series of three solvents of decreasing polarity (ammonium phosphate buffer, 10% methanol, and 50% methanol) was designed. By this procedure DHPS was detected in Escherichia coli and Pneumocystis carinii with specific activities of 450 and 14 U/mg, respectively. A comparison of the effects of five sulfa drugs on P. carinii DHPS activity revealed that dapsone is the most potent of these drugs.Pneumocystis carinii is the major life-threatening opportunistic agent in patients with acquired immunodeficiency syndrome (AIDS) and affects other immunocompromised patients. Approximately 60% of all patients with AIDS suffer from P. carinii pneumonia, and up to 40% of the episodes prove fatal (10).Patients with P. carinii pneumonia are frequently treated with antifolates (inhibitors of tetrahydrofolate biosynthesis) such as bactrim (a combination of trimethoprim and sulfamethoxazole) (6, 7), dapsone (6, 13), and trimetrexate (1). The ability of P. carinii to synthesize tetrahydrofolate de novo has been reported recently (9).Two enzymes involved in the biosynthesis of tetrahydrofolate that are important as targets of antifolates are dihydropteroate synthetase (DHPS) and dihydrofolate reductase. DHPS catalyzes the formation of dihydropteroate (DHP) from p-aminobenzoic acid (pAB) and 6-hydroxymethyldihydropterin pyrophosphate (MDHP-PP). Dihydrofolate reductase catalyzes the formation of tetrahydrofolate from dihydrofolate. Sulfones and sulfonamides act as competitive inhibitors of DHPS substrates, while drugs such as trimethoprim and pyrimethamine act as inhibitors of dihydrofolate reductase substrates. In this study we measured the inhibitory actions of several sulfonamides and sulfones on the parasite associated with DHPS using a new high-pressure liquid chromatography (HPLC) enzyme assay. MATERIALS AND METHODSGrowth and isolation of organisms. Male Sprague-Dawley rats (weight, 150 to 200 g) were fed ad libitum and administered tetracycline in their drinking water (1 g/liter). Subcutaneous injections of cortisone acetate (25 mg) were given twice a week. After 4 to 6 weeks of injections, those rats which showed signs of rapid breathing, blood in their noses, weight loss, and hair loss were used in the experiments.P. carinii was isolated by a modification of the method of Gradus and Ivey (4). Each rat was sacrificed by cervical dislocation. Their lungs were then quickly removed and kept at 4°C. Touch preparations were made from the lungs and stained with toluidine blue (3) bacteria were evident. Subsequent inoculation of the lung homogenate onto Luria broth agar plates confirmed the absence of contaminants. The lungs were homogenized by repeated passage through a 60-gauge wire screen. The resulting suspension was centrifuged at 2,000 x g for 10 min. The supernatant was discarded, and the pellet was suspended in phosphate-buffered saline. The suspensio...

Injury to the spinal cord often results in abnormal lateral curvature of the spine, or scoliosis, that is associated with neuromuscular weakness. The lateral curvature of the spine is thought to be a consequence of insufficient or asymmetrical loading of the vertebrae. To study neuromuscular scoliosis, an animal model of spinal cord injury was used in which the spinal cord was partially (3/4) transected, with the left lateral columns left intact. Partial transection of the spinal cord in the rat caused scoliosis that was maximal four to five vertebrae distal to the lesion site. As in previous experiments involving unilateral spinal cord lesions, the scoliotic curves were convex on the weakened side. Subtotal transection at T5 or T11 resulted in lateral displacement of vertebrae T9-T12 or L2-L5, respectively, of up to 11 mm. Interestingly, this vertebral displacement is greatly reduced by clenbuterol, a beta2-adrenoceptor agonist that has been found to retard loss of muscle contractility and bone mineralization due to denervation. Together these results suggest that stimulation of beta2-receptors opposes vertebral unloading due to neuromuscular weakness and thereby acts as a countermeasure to scoliosis.