Continuous axenic cultivation of Pneumocystis carinii

Continuous axenic culture of Pneumocystis carinii has been achieved. A culture vessel is used that allows for frequent medium exchange without disturbance of organisms that grow attached to a collagen-coated porous membrane. The growth medium is based on Minimal Essential Medium with Earle’s salt supplemented with S-adenosyl-l-methionine, putrescine, ferric pyrophosphate, N-acetyl glucosamine, putrescine, p-aminobenzoic acid, l-cysteine and l-glutamine, and horse serum. Incubation is in room air at 31°C. The pH of the medium begins at 8.8 and rises to ≈9 as the cells grow. Doubling times calculated from growth curves obtained from cultures inoculated at moderate densities ranged from 35 to 65 hours. With a low-density inoculum, the doubling time is reduced to 19 hours. The morphology of cultured organisms in stained smears and in transmission electron micrographs is that of P. carinii, and P. carinii-specific mAbs label the cultured material. Cultured organisms are infective for immunosuppressed rats and can be stored frozen and used to reinitiate culture.

Association of Ferredoxin-NADP Oxidoreductase with the Chloroplastic Pyridine Nucleotide Dehydrogenase Complex in Barley Leaves1

Barley (Hordeum vulgare L.) leaves were used to isolate and characterize the chloroplast NAD(P)H dehydrogenase complex. The stroma fraction and the thylakoid fraction solubilized with sodium deoxycholate were analyzed by native polyacrylamide gel electrophoresis, and the enzymes detected with NADH and nitroblue tetrazolium were electroeluted. The enzymes electroeluted from band S from the stroma fraction and from bands T1 (ET1) and T2 from the thylakoid fraction solubilized with sodium deoxycholate had ferredoxin-NADP oxidoreductase (FNR; EC 1.18.1.2) and NAD(P)H-FeCN oxidoreductase (NAD[P]H-FeCNR) activities. Their NADPH-FeCNR activities were inhibited by 2′-monophosphoadenosine-5′-diphosphoribose and by enzyme incubation with p-chloromercuriphenylsulfonic acid (p-CMPS), NADPH, and p-CMPS plus NADPH. They presented Michaelis constant NADPH values that were similar to those of FNRs from several sources. Their NADH-FeCNR activities, however, were not inhibited by 2′-monophosphoadenosine-5′-diphosphoribose but were weakly inhibited by enzyme incubation with NADH, p-CMPS, and p-CMPS plus NADH. We found that only ET1 contained two polypeptides of 29 and 35 kD, which reacted with the antibodies raised against the mitochondrial complex I TYKY subunit and the chloroplast ndhA gene product, respectively. However, all three enzymes contained two polypeptides of 35 and 53 kD, which reacted with the antibodies raised against barley FNR and the NADH-binding 51-kD polypeptide of the mitochondrial complex I, respectively. The results suggest that ET1 is the FNR-containing thylakoidal NAD(P)H dehydrogenase complex.