Aminoglycoside antibiotics preferentially increase permeability in phosphoinositide-containing membranes: a study with carboxyfluorescein in liposomes

The rate of release from multilamellar liposomes of the fluorescent probe carboxyfluorescein was determined as a measure of membrane permeability. Liposomes of phosphatidylcholine and different anionic phospholipids were incubated with low (1 microM) and high (3 mM) concentrations of calcium in the absence or presence of aminoglycoside antibiotics. The leakage of carboxyfluorescein into the medium was not caused by liposomal fusion as no vesicle fusion was observed in experiments with terbium and dipicolinic acid-loaded liposomes. The basal rate of carboxyfluorescein release (in the absence or presence of 1 microM calcium) from all types of liposomes ranged from 0.1 to 0.3% of trapped carboxyfluorescein per hour. The presence of 3 mM calcium caused the greatest increase in the rate of carboxyfluorescein release (about 9-fold) in liposomes containing phosphatidylinositol 4,5-bisphosphate (PIP2) whereas liposomes containing the other anionic phospholipids (phosphatidylserine, phosphatidylinositol and phosphatidylinositol 4-phosphate) showed an approximate 5-fold increase. In the presence of 1 microM calcium, the aminoglycosides neomycin and gentamicin also increased the rate of carboxyfluorescein release, with PIP2-containing liposomes showing a 3-5-times greater response than the other liposomes, releasing up to 4.6% of trapped carboxyfluorescein per hour. This drug-induced release was dose-dependent and antagonized by calcium. In the presence of 3 mM calcium, 0.1 mM gentamicin or neomycin were ineffective while the drug at 1 mM affected carboxyfluorescein release from PIP2-liposomes only. The aminoglycoside antibiotics, neomycin, gentamicin, tobramycin, kanamycin, amikacin, netilmicin, as well as neamine and spectinomycin (all at 0.1 mM) showed a graded effect on the rate of carboxyfluorescein release from PIP2-containing vesicles in the presence of 0.1 mM calcium. The magnitude of the effect correlated well with the ototoxicity of the drugs previously determined directly in cochlear perfusions in the guinea pig. The study demonstrates that aminoglycoside antibiotics are capable of altering membrane permeabilities and that this effect is most pronounced if PIP2 is present in the bilayers. The excellent correlation between this membrane action and the in-situ toxicity of the drugs further establishes the specific role of PIP2 in the molecular mechanism of aminoglycoside-induced hearing loss. Moreover, it confirms the usefulness of such physicochemical models for the screening and prediction of aminoglycoside toxicity.     

Cytologic findings in progressive multifocal leukoencephalopathy. Report of two cases

Cytologic preparations from two cases of progressive multifocal leukoencephalopathy (PML) were obtained by stereotactically guided needle biopsies using computerized tomograms of the brain. Case 1 was a 32-year-old man with an acquired immunodeficiency syndrome-related complex. Case 2 was a 71-year-old man with chronic lymphocytic leukemia. Smears showed moderate cellularity, consisting of moderately to markedly atypical cells with enlarged hyperchromatic nuclei. The chromatin pattern showed smudging, with or without clumping, similar in pattern to the human polyomavirus-infected "decoy" cells seen in urine cytology. Nuclei were predominantly round to oval, smoothly contoured and often stripped of cytoplasm. Occasional bizarre lobulated or multinucleated forms were seen. Some atypical cells had abundant cytoplasm exhibiting stellate projections. Histologic sections of the biopsy material confirmed the diagnosis in each case. In both cases, electron microscopy demonstrated intranuclear polyoma-type virus particles. The present findings suggest that PML should be considered in the differential diagnosis of marked cytologic atypia in brain aspirates from immunocompromised patients.     

The arsenical ATPase efflux pump mediates tellurite resistance.

The ars operon of the resistance plasmid R773 was found to produce moderate levels of resistance to tellurite. A MIC of 64 micrograms of TeO3(2-) per ml was found for Escherichia coli cells harboring plasmids which contained all three of the structural genes (arsA, arsB, and arsC) of the anion-translocating ATPase. MICs specified by plasmids carrying only one or two structural elements or the cloning vector alone were 2 to 4 micrograms/ml. The rate of TeO3(2-) uptake was found to be on the order of 55% less for cultures containing the resistance plasmids.     

Inter- and intracellular distribution of amino acids and other metabolites in maize (Zea mays L.) leaves

In illuminated maize (Zea mays L.) leaves, the distribution of triose phosphates, 3-phosphoglycerate, malate and various amino acids between the chloroplastic and the extrachloroplastic compartments of mesophyll and bundle-sheath cells, and the total vacuolar fraction of the leaves, was determined by a combination of previously published methods, for separating mesophyll from bundle-sheath material, and for nonaqueous subcellular fractionation. The results show that the triose phosphate/3-phosphoglycerate ratio in the extrachloroplastic fraction of the mesophyll cells is about 20-fold higher than in the bundle-sheath cells, which is in accordance with a triose phosphate/phosphoglycerate shuttle postulated previously. Whereas the vacuolar compartment was shown to contain most of the cellular malate, amino acids were found to be almost absent from this compartment. The amino-acid pattern in the extrachloroplastic fraction of the bundle-sheath cells largely resembled the pattern in whole leaves. These results show that for future studies the analysis of amino-acid contents in whole maize leaves can be used as a measure for the amino-acid levels in the cytosol of bundle-sheath cells.Abbreviations BS bundle sheath - Chl chlorophyll - Man -mannosidase - ME malic enzyme - MDH malate dehydrogenase - MS mesophyll - PEPCase phosphoenolpyruvate carboxylase - PGA 3-phosphoglycerate - trioseP triose phosphate This work was supported by the Bundesminister für Forschung und Technologie.     

Cross-linking of U1 snRNA using nitrogen mustard. Evidence for higher order structure.

Secondary structures for all five spliceosomal small nuclear (sn) RNAs (U1, U2, U4, U5, and U6 snRNAs) have been derived from phylogenetic, biochemical, and genetic data, but tertiary structure information has been more difficult to obtain. Here we have used the general cross-linking reagent nitrogen mustard (bis-(2-chloroethyl)methylamine) to explore the tertiary conformation of naked U1 snRNA. We detected two intramolecularly cross-linked U1 species (X-U1#1 and X-U1#2) after cross-linking of deproteinized HeLa nuclear extract. We determined the cross-linked sites and found that X-U1#1 is cross-linked between the C82-A85 and U129, while X-U1#2 is cross-linked between U105-G108 and A118. We then tested the ability of these two cross-linked species to bind small nuclear ribonucleo-protein (snRNP) proteins in vitro (in HeLa nuclear extract or S100) and in vivo (in Xenopus oocytes). Both X-U1#1 and X-U1#2 were found to reconstitute efficiently in vitro and in vivo, as judged by immunoprecipitation with antibodies specific for Sm and U1-specific proteins. Our data suggest that (i) the Sm-binding site lies on the surface of the native U1 snRNP, since the cross-link in X-U1#1 involves the Sm-binding site but does not block snRNP assembly, and (ii) U1 snRNA may adopt the correct tertiary conformation even in the absence of U1 snRNP proteins.     

Cross-linking of U2 snRNA using nitrogen mustard. Evidence for higher order structure.

Nuclear mRNA precursors are spliced by a large macromolecular complex called the spliceosome which contains, in most eucaryotes, five small nuclear RNAs (snRNAs) each in the form of a small ribonucleoprotein particle (the U1, U2, U5, and U4/U6 snRNPs). Although secondary structures have been derived for all five spliceosomal snRNAs based on phylogenetic, biochemical, and genetic data, little tertiary structure information is available. Here we use the general cross-linking reagent nitrogen mustard [bis-(2-chloroethyl)methylamine] to detect tertiary interactions within U2 snRNA. After the cross-linking of deproteinized HeLa nuclear extract, two intramolecularly cross-linked U2 species with anomalous electrophoretic mobility can be detected (X-U2#1 and X-U2#2). The 3' and 5' boundaries of each cross-link were determined by rapid enzymatic RNA sequencing of end-labeled RNA. X-U2#1 is cross-linked between the region U41-U55 and G105 or G106, X-U2#2 between U53 and G97 or G98. We then tested the ability of the two cross-linked species to bind snRNP proteins in vitro (in nuclear extract or S100) and in vivo (in Xenopus oocytes). X-U2#2 reconstituted efficiently both in vitro and in vivo but X-U2#1 did not, as judged by immunoprecipitation with antibodies specific for Sm- and U2-specific proteins. Since the cross-link in X-U2#2 involves the Sm binding site but does not block snRNP assembly, our data strongly suggest that the Sm binding site lies on the surface of the native snRNP.     

21-Aminosteroids Interact with the Dopamine Transporter to Protect Against 1-Methyl-4-Phenylpyridinium-Induced Neurotoxicity

U-78518F, a 21-aminosteroid from the novel family of lipid peroxidation inhibitors (lazaroids), increased survival of dopamine (DA) neurons in mesencephalic cell cultures incubated with the neurotoxin 1-methyl-4-phenylpyridinium (MPP+). Protection against DA neuron death occurred with increasing concentrations of U-78518F up to 30 microM. Non-specific toxicity produced with higher concentrations of MPP+ was not affected by the lazaroid. U-78518F inhibited cellular uptake of [3H]MPP+ and [3H]DA, but not that of gamma-[3H]aminobutyric acid. In human striatal membrane preparations, U-78518F competed with [3H]mazindol for binding to the DA transporter, with a calculated Ki value of 10 microM. Two of four lazaroids tested inhibited [3H]DA uptake in the cell culture system. The protective effects of 21-aminosteroids in MPP(+)-induced neurotoxicity are, in part, a function of the interaction of these agents with the DA transporter.