Trypanosoma brucei gambiense: growth in vitro of bloodstream forms inhibited by dichlorodiammineplatinum (II) compounds and hypolipidemic drugs

Infectious bloodstream forms of Trypanosoma brucei gambiense were grown in microcultures of murine bone marrow cells in 96-well tissue culture plates. Limiting dilution studies showed that fewer than 10 cultured trypanosomes developed into populations of about 5 X 10(4) parasites per well in a week. Bloodstream parasites were reisolated with high efficiency from mice infected with cultured parasites; fewer than 10 bloodstream parasites successfully established a trypanosome population in a microculture. Both the cis and trans isomers of dichlorodiammineplatinum (II) (cisplatin and transplatin) and a hypolipidemic agent, Wy 14643, were found to have activity against T. b. gambiense growing in microcultures. The minimum concentration of drug necessary to completely eliminate parasites from microcultures was 4 microM for cisplatin, 40 microM for transplatin, and 500 microM for Wy 14643. A preformed complex of cisplatin and bovine serum albumin and another hypolipidemic agent, chlofibric acid, were inactive. This culture system should be useful for rapid screening of large numbers of compounds for trypanocidal activity.     

Conformation-specific precipitation of human α2-macroglobulin by divalent zinc or calf thymus histone H3

Highly purified native α₂-macroglobulin (α₂M), α₂M-trypsin, and α₂M-methylamine were compared in experiments designed to study protein precipitation. Significant turbidity developed within 30 min in solutions containing histone H3 and either α₂M-methylamine or α₂M-trypsin, as determined by absorbance at λ = 550 nm. No turbidity was detected in solutions that contained histone H3 and native α₂M or histone H3 alone. Experiments with radioiodinated histone H3 or radioiodinated proteinase inhibitor confirmed that both the H3 and the α₂M “fast” forms (α₂M-methylamine, α₂M-trypsin) were present in the precipitates generated. As much as 70% of the ¹²⁵I-α₂M-methylamine was recovered in the precipitate after incubation with a 120-fold molar excess of H3 (concentration of α₂M-methylamine, 0.28 μm). The ratio of histone to proteinase inhibitor by weight in the precipitate was approximately two. Under comparable conditions, somewhat less α₂M-trypsin precipitated from solutions containing H3 than did α₂M-methylamine; however, inactivation of the α₂M-trypsin with phenylmethylsulfonyl fluoride prior to incubation increased the level of precipitation significantly. Solutions containing poly-l-lysine (M_r ~ 13,000) instead of histone did not form precipitates with any of the forms of α₂M studied. In a second set of experiments, radioiodinated native α₂M, α₂M-trypsin, and α₂M-methylamine were incubated in solutions containing ZnCl₂, BaCl₂, CdCl₂, CuSO₄, MgCl₂, or NiCl₂ (concentration of divalent cation between 5 μm and 1.0 mm). Native α₂M was soluble in all of these salts. By contrast, α₂M-methylamine and α₂M-trypsin precipitated extensively from solutions containing greater than 100 μm ZnCl₂. Precipitation was greater than 90% complete at 1 mm ZnCl₂. A similar effect was not observed with any of the other divalent cations.