Toxic effects of antimalarial drugs in Paramecium : role of calcium channels

The antimalarial drugs, quinacrine, quinine and mefloquine, as well as the structurally-similar compound, W-7, inhibit calcium-dependent backward swimming and calcium currents in Paramecium calkinsi. These drugs are also toxic to paramecia at high concentrations. Therefore, one site of toxic action of the drugs may be the calcium channel. To test this hypothesis, the toxicity of the antimalarials and W-7 was compared in paramecia with and without calcium channels. Since calcium channels are located on the cilia, calcium channels were removed from the paramecia by deciliating the cells. Deciliated cells were found to be less susceptible to the lethal effects of the antimalarials and W-7 than their ciliated counterparts. Moreover, Pawns, mutants of P. tetraurelia that possess cilia but lack functional calcium channels, were also less susceptible to the antimalarials than wild-type cells. Thus, calcium channels may be one site of toxic action of the antimalarial drugs in paramecia and perhaps in other protists. Accepted: 27 December 1996     

Fluid and ion transport in corneal endothelium: insensitivity to modulators of Na+-K+-2Cl- cotransport

The role ofNa⁺-K⁺-2Clcotransport in ion and fluid transport of the corneal endothelium wasexamined by measuring changes in corneal hydration and uptake of⁸⁶Rb by the endothelial celllayer. Isolated, intact rabbit corneas maintain normal hydration whenthey are superfused at the endothelial surface with bicarbonate()-Ringer solutions as aresult of equilibrium between active ion and fluid transport out of thestromal tissue and leak of fluid into stromal tissue from the aqueoushumor. Furosemide and bumetanide did not alter this equilibrium whenthey were added to the superfusion medium. Uptake of⁸⁶Rb by the endothelium of theincubated cornea was increased 25% by bumetanide, but uptake in thepresence of ouabain (70% less than that of controls) was not changedby bumetanide. In Na⁺-free medium,uptake of ⁸⁶Rb was reduced by58%, but it was unchanged inCl-free medium. CalyculinA, a protein phosphatase inhibitor and activator ofNa⁺-K⁺-Clcotransport, was without effect on⁸⁶Rb uptake. Hypertonicity (345 mosmol/kg) increased uptake slightly, whereas hypotonicity (226 mosmol/kg) caused a 33% decrease. Neither of these changes wassignificantly different when bumetanide was present in the media. It isconcluded thatNa⁺-K⁺-2Clcotransporter activity is not exhibited by the in situ corneal endothelium and does not play a role in the ion and fluid transport ofthis cell layer. Its presence in cultured endothelial cells may reflectthe reported importance of this protein in growth, proliferation, anddifferentiation.

     

Functional Expression of Drosophila para Sodium Channels : Modulation by the Membrane Protein TipE and Toxin Pharmacology

The Drosophila para sodium channel α subunit was expressed in Xenopus oocytes alone and in combination with tipE, a putative Drosophila sodium channel accessory subunit. Coexpression of tipE with para results in elevated levels of sodium currents and accelerated current decay. Para/TipE sodium channels have biophysical and pharmacological properties similar to those of native channels. However, the pharmacology of these channels differs from that of vertebrate sodium channels: (a) toxin II from Anemonia sulcata, which slows inactivation, binds to Para and some mammalian sodium channels with similar affinity (K _d ≅ 10 nM), but this toxin causes a 100-fold greater decrease in the rate of inactivation of Para/TipE than of mammalian channels; (b) Para sodium channels are >10-fold more sensitive to block by tetrodotoxin; and (c) modification by the pyrethroid insecticide permethrin is >100-fold more potent for Para than for rat brain type IIA sodium channels. Our results suggest that the selective toxicity of pyrethroid insecticides is due at least in part to the greater affinity of pyrethroids for insect sodium channels than for mammalian sodium channels.     

Role of oxygen limitation and nitrate metabolism in the nitrate inhibition of nitrogen fixation by pea

The impact of nitrate (5–15 m M , 2 to 7 days) on nitrogenase activity and nodule-oxygen limitation was investigated in nodulated, 21-day-old plants of a near-isogenic nitrate reductase-deficient pea mutant (A3171) and its wild-type parent ( Pisum sativum L. cv. Juneau). Within 2 days, 10 or 15 m M nitrate, but not 5 m M nitrate, inhibited the apparent nitrogenase activity (measured as in situ hydrogen evolution from nodules of intact plants) of wild-type plants; none of these nitrate levels inhibited the apparent nitrogenase activity of A3171 plants. Nodule-oxygen limitation, measured as the ratio of total nitrogenase activity to potential nitrogenase activity, was increased in both wild-type and A3171 plants by all nitrate treatments. By 3 to 4 days the apparent nitrogenase activity of A3171 and wild-type plants supplied with 5 m M nitrate declined to 53 to 69% of control plants not receiving nitrate. By 6 to 7 days the apparent nitrogenase activity of A3171 plants was similar to the control value whereas that of the wild-type plants continued to decline. From 3 to 7 days, no significant differences in nodule-oxygen limitation were observed between the nitrate (5 m M ) and control treatments. The results are interpreted as evidence for separate mechanisms in the initial (O₂ limitation) and longer-term (nitrate metabolism) effects of nitrate on nitrogen fixation by effectively nodulated pea.     

The relative significance of mechanisms of antigenic variation in African trypanosomes

The large number of genes involved in antigenic variation in African trypanosomes has been the focus of a wide literature that describes an almost bewildering array of mechanisms for their differential activation. To the outsider searching for an underlying strategy for antigenic variation, this can appear as a rather disordered and confusing picture. Here, David Barry argues that an understanding of which mechanisms are significant, which ones are primarily inconsequential and which ones perhaps even arise from overdependence on laboratory models, might be achieved by turning attention to trypanosomes that have not undergone adaptation in laboratory conditions. Application of such an approach has led to a proposal for a main mechanism for antigenic variation.     

Partial alanine scan of mast cell degranulating peptide (MCD): importance of the histidine- and arginine residues.

The influence of the two histidine and two arginine residues of mast cell degranulating peptide (MCD) in activity and binding was studied by replacing these amino acids in the MCD sequence with L-alanine. Their histamine releasing activity was determined on rat peritoneal mast cells. Their binding affinity to the FcepsilonRIalpha binding subunit of the human mast cell receptor protein, was carried out using fluorescence polarization. The histamine assay showed that replacement of His13 by Ala o ccurred without loss of activity compared with the activity of MCD. Alanine substitutions for Arg7 and His8 resulted in an approximately 40 fold increase, and for Arg16 in a 14-fold increase in histamine-releasing activity of MCD. The binding affinities of the analogs were tested by competitive displacement of bound fluorescent MCD peptide from the FcepsilonRIalpha binding protein of the mast cell receptor by the Ala analogs using fluorescence polarization. The analogs Ala8 (for His) and Ala16 (for Arg) showed the same binding affinities as MCD, whereas analog Ala7 (for Arg) and analog Ala13 (for His) showed slightly better binding affinity than the parent compound. This study showed that the introduction of alanine residues in these positions resulted in MCD agonists of diverse potency. These findings will be useful in further MCD structure-activity studies.     

The specificity of methyl transferases involved in trans mycolic acid biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Trans mycolic acid content is directly related to cell wall fluidity and permeability in mycobacteria. Carbon-13 NMR spectroscopy of mycolic acids isolated from Mycobacterium tuberculosis (MTB) and Mycobacterium smegmatis (MSM) fed 13C-labeled precursor molecules was used to probe the biosynthetic pathways that modify mycolic acids. Heteronuclear correlation spectroscopy (HMQC) of ketomycolic acid from MTB allowed assignment of the complete 13C-NMR spectrum. Incorporation patterns from [1-13C]-acetate and [2-13C]-acetate feeding experiments suggested that the mero chain and alpha branch of mycolic acids are both synthesized by standard fatty acid biosynthetic reactions. [13C-methyl]-L-methionine was used to specifically label carbon atoms derived from the action of the methyl transferases involved in meromycolate modification. To enrich for trans mycolic acids a strain of MTB overexpressing the mma1 gene was labeled. Carbon-carbon coupling was observed in mycolate samples doubly labeled with 13C-acetate and [13C-methyl]-L-methionine and this information was used to assess positional specificity of methyl transfer. In MTB such methyl groups were found to occur exclusively on carbons derived from the 2 position of acetate, while in MSM they occurred only on carbons derived from the 1 position. These results suggest that the MSM methyltransferase MMAS-1 operates in an inverted manner to that of MTB.