Cyclophilins of a novel subfamily interact with SNW/SKIP coregulator in Dictyostelium discoideum and Schizosaccharomyces pombe.

We screened the Dictyostelium discoideum two-hybrid cDNA library with the SNW/SKIP transcription coregulator SnwA and identified a novel cyclophilin CypE. Independently, the Schizosaccharomyces pombe cDNA library was screened with the SnwA ortholog Snw1 and the ortholog of CypE (named Cyp2) was found. Both cyclophilins bind the respective SNW protein in their autologous systems. The interaction was localized to the N-terminal part of SnwA as well as of Snw1. CypE was confirmed in vitro to be a cyclosporin A-sensitive peptidyl-prolyl cis-trans isomerase. Remarkably, both SNW proteins bind the cyclophilins in a cyclosporin A independent manner, possibly serving as adaptors for these novel isomerases. These results are the first characterization of the members of a novel cyclophilin subfamily, which includes the human CGI-124/PPIL1 protein.

Molecular characterization of a calmodulin-like dictyostelium protein CalB.

A gene named calB was cloned and characterized in Dictyostelium. A relationship to calmodulin (CaM) is suggested by sequence identity (50%), similar exon-intron structure and cross-reactivity with anti-CaM sera. The level of calB mRNA is developmentally regulated with maxima during aggregation and in spores. CalB null cells grow normally, develop and produce viable spores. We demonstrated the capacity of tagged CalB to bind Ca(2+) using the (45)Ca(2+) overlay assay and showed that its mobility on SDS-PAGE is dependent on Ca(2+)/EGTA pretreatment.

The homolog of chromatin binding protein Bx42 identified in Dictyostelium.

We identified in Dictyostelium a gene snwA containing a region of similarity to SH2 domains of higher eukaryotes. snwA is homologous to a novel human gene SNW1 and to Bx42 from Drosophila melanogaster, a gene coding for a chromatin binding protein responsive to 20-OH-ecdysone. snwA has one mRNA transcript of an approximate size of 2.5 kb.

Metabolic differentiation of bloodstream forms of Trypanosoma brucei brucei into procyclic forms in hemin-depleted medium and in the presence of respiratory inhibitors.

Bloodforms of Trypanosoma brucei brucei (STIB 247) differentiated in vitro into procyclic forms as described in the accompanying paper (Markos et al. 1989). The importance of the respiratory chain for the process was tested by the inhibition of its development (omission of hemin from the medium) or function (respiratory inhibitors). In the absence of hemin, all enzyme markers of the procyclic state, except for hemoproteins, developed to 50-70 per cent of control values. The presence of hemin is therefore not essential for the onset of differentiation, although the process cannot be completed under hemin limitation. Addition of 1 mumole.dm-3 KCN, 10 mumole.dm-3 antimycin A, or 100 mumole.dm-3 salicyl hydroxamate (SHAM) did not block the differentiation, although it proceeded at a slower rate. The development of the inner mitochondrial membrane markers--succinate: cytochrome c reductase, and NADH: cytochrome c reductase--was strongly inhibited by KCN or antimycin. None of these inhibitors had a significant effect on the activity of procyclic state marker--glycosomal malate dehydrogenase.

Metabolic differentiation of bloodstream forms of Trypanosoma brucei brucei into procyclic forms. Effect of hydroxyurea, arabinosyl adenine, and serum omission.

Bloodforms of Trypanosoma brucei brucei STIB 247 taken from rats and containing more than 80 per cent short stumpy forms, differentiated in vitro to procyclic forms in medium SDM 79 (Brun and Schönenberger 1979), enriched with 3 mmol.dm-3 cis-aconitate. Cell division was abolished by the addition of hydroxyurea (200 micrograms.ml-1) or arabinosyl adenine (20 micrograms.ml-1 to the cultivation medium, or by the omission of serum from the medium. The ultrastructure of exponentially growing controls was rearranged within 24 h. The endogenous respiration and the respiration stimulated by proline, succinate, and 2-oxoglutarate were detectable within 12 h; after 48 h the respiration rates were comparable to those found in the established procyclic forms. After 12 h the respiration was inhibited by 200 mumol.dm-3 KCN, and by 20 mumol.dm-3 antimycin to the extent found in procyclic forms. Hydroxyurea did not significantly affect respiration. Activities of procyclic-stage enzyme markers malate dehydrogenase, threonine dehydrogenase, succinate: cytochrome c reductase, and NADH: cytochrome c reductase rose within 48 h of differentiation to values which were close to those found in established procyclic forms. The activity of glutamate dehydrogenase (NAD-specific), however, was only 1/3 of that in the procyclics, and no citrate synthase was detected in differentiating culture. Glycosomal malate dehydrogenase was detected after 6 h. In the presence of hydroxyurea or arabinosyl adenine, or in the absence of serum, respiration rates, marker enzyme activities, and glycosomal malate dehydrogenase developed to the extent comparable to the untreated controls. The results suggest that it is possible to separate the process of differentiation from cell proliferation. Cell division is not a necessary prerequisite of differentiation.

Inactivation of bacteriophages with cis-platinum(II) diamminedichloride.

The ability of the cis-plantinum(II) diamminedichloride and its hydrolytic products to inactive DNA bacteriophages was examined on the models T2, T4, T4BO1, T3, and lambda. The inactivation of all bacteriophages under study increases gradually during the first 40-90 min of the action of neutral cis-Pt(II) and later passes into an exponential phase. The extent of the region of slower inactviation is larger for osmotically sensitive strains T2 and T4. Inactivation with the hydrolytic products for cis-Pt(II) proceeds exponentially starting from the very beginning and their inactivating effect is higher by 40-80 times than for a comparable concentration of the original complex. The extent of inactivation is not affected with the HCR marker of the host bacteria. The sensitivity to cis-Pt(II) is higher for bacteriophages with a head permeable to salts. An additional inactivation ("after-effect") was observed after dilution of the complex; it can be removed by adding S-aminoisothiuronium dihydrobromide (AET). The results obtained are in good accord with the assumption that inactivation is due to the hydrolytic products arising in the head of bacteriophage.