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Biomedical subjects

J Lenard

Publications and source records attributed to J Lenard.

At least 55 records · Page 3Linked to original sources
PubMed1987

Effects of growth state and amines on cytoplasmic and vacuolar pH, phosphate and polyphosphate levels in Saccharomyces cerevisiae: a 31P-nuclear magnetic resonance study.

The vacuoles of logarithmic and stationary stage cells were compared by 31P-NMR with regard to pH, orthophosphate (Pi) content and average size of polyphosphate. The vacuoles of stationary cells had lower pH, higher Pi content, and polyphosphates of longer average chain length, although total polyphosphate content was about the same as in logarithmic cells. The lower vacuolar pH in stationary cells was the major cause of a larger cytoplasmic-vacuolar pH gradient. Addition of NH4Cl, (NH4)2SO4, methylamine or amantadine at pH 8 to cells in either stage caused an increase in both cytoplasmic and vacuolar pH, with little or no change in the cytoplasmic-vacuolar pH gradient. However, the administration of ammonium salts to the cells at pH 8.0 resulted in rapid hydrolysis of the intravacuolar polyphosphate to tripolyphosphate and Pi, with attendant redistribution of Pi between the vacuolar and cytoplasmic compartments.

Amantadine↗
PubMed1987

Estimation by radiation inactivation of the size of functional units governing Sendai and influenza virus fusion.

The target sizes associated with fusion and hemolysis carried out by Sendai virus envelope glycoproteins were determined by radiation inactivation analysis. The target size for influenza virus mediated fusion with erythrocyte ghosts at pH 5.0 was also determined for comparison; a value of 57 +/- 15 kDa was found, indistinguishable from that reported previously for influenza-mediated fusion of cardiolipin liposomes [Gibson, S., Jung, C. Y., Takahashi, M., & Lenard, J. (1986) Biochemistry 25, 6264-6268]. Sendai-mediated fusion with erythrocyte ghosts at pH 7.0 was likewise inactivated exponentially with increasing radiation dose, yielding a target size of 60 +/- 6 kDa, a value consistent with the molecular weight of a single F-protein molecule. The inactivation curve for Sendai-mediated fusion with cardiolipin liposomes at pH 7.0, however, was more complex. Assuming a "multiple target-single hit" model, the target consisted of 2-3 units of ca. 60 kDa each. A similar target was seen if the liposomes contained 10% gangliosides or if the reaction was measured at pH 5.0, suggesting that fusion occurred by the same mechanism at high and low pH. A target size of 261 +/- 48 kDa was found for Sendai-induced hemolysis, in contrast with influenza, which had a more complex target size for this activity (Gibson et al., 1986). Sendai virus fusion thus occurs by different mechanisms depending upon the nature of the target membrane, since it is mediated by different functional units. Hemolysis is mediated by a functional unit different from that associated with erythrocyte ghost fusion or with cardiolipin liposome fusion.

Animals↗
PubMed1987

Inactivation of the RNA polymerase of vesicular stomatitis virus by N-ethylmaleimide and protection by nucleoside triphosphates. Evidence for a second ATP binding site on L protein.

The purified RNA polymerase complex of vesicular stomatitis virus required added thiols for maximal activity, whereas polymerase activity from whole disrupted virions did not. Maximal activity of the purified polymerase complex required greater than or equal to 1 mM added dithiothreitol. The polymerase was inactivated by N-ethylmaleimide (NEM) at 0 degree C, with k2 = 528 +/- 26 M-1 min-1. Activity was recovered by addition of L protein, but not N or NS, to the NEM-inactivated complex, indicating that the NEM-sensitive group was present on the L protein. Nucleoside triphosphates protected the enzyme against inactivation by N-ethylmaleimide. ATP was most effective, with KD = 0.58 +/- 0.07 mM, a value close to the Km of ATP reported previously for initiation of RNA synthesis. dATP was nearly as effective, and GTP was slightly less effective than ATP. Non-hydrolyzable analogs of ATP protected weakly, whereas ADP and pyrimidine triphosphates gave very poor, but still measurable, protection. The ATP binding site thus identified differs from the protein kinase-associated ATP binding site identified on L protein by Sanchez et al. (Sanchez, A., De, B.P., and Banerjee, A. K. (1985) J. Gen. Virol. 66, 1025-1036) in having a substantially lower affinity for ATP. Two putative ATP binding sites were identified in the L protein amino acid sequence, but none were found in the N or NS sequences.

Animals↗
PubMed1987

The role of lysosomal enzymes in killing of mammalian cells by the lysosomotropic detergent N-dodecylimidazole.

The sensitivity of cultured human and hamster fibroblast cells to killing by the lysosomotropic detergent N-dodecylimidazole (C12-Im) was investigated as a function of cellular levels of general lysosomal hydrolase activity, and specifically of cysteine cathepsin activity. Fibroblasts from patients with mucolipidosis II (I-cell disease) lack mannose-6-phosphate-containing proteins, and therefore possess only 10-15% of the normal level of most lysosomal hydrolases. I-cell fibroblasts are about one-half as sensitive to killing by C12-Im as are normal human fibroblasts. Overall lysosomal enzyme levels of CHO cells were experimentally manipulated in several ways without affecting cell viability: Growth in the presence of 10 mM ammonium chloride resulted in a gradual decrease in lysosomal enzyme content to 10-20% of control values within 3 d. Subsequent removal of ammonium chloride from the growth medium resulted in an increase in lysosomal enzymes, to approximately 125% of control values within 24 h. Treatment with 80 mM sucrose caused extensive vacuolization within 2 h; lysosomal enzyme levels remained at control levels for at least 6 h, but increased 15-fold after 24 h of treatment. Treatment with concanavalin A (50 micrograms/ml) also caused rapid (within 2 h) vacuolation with a sevenfold rise in lysosomal enzyme levels occurring only after 24 h. The sensitivity of these experimentally manipulated cells to killing by C12-Im always paralleled the measured intracellular lysosomal enzyme levels: lower levels were associated with decreased sensitivity while higher levels were associated with increased sensitivity, regardless of the degree of vacuolization of the cells. The cytotoxicity of the cysteine proteases (chiefly cathepsin L in our cells) was tested by inactivating them with the irreversible inhibitor E-64 (100 micrograms/ml). Cell viability, protein levels, and other lysosomal enzymes were unaffected, but cysteine cathepsin activity was reduced to less than 20% of control values. E-64-treated cells were almost completely resistant to C12-Im treatment, although lysosomal disruption appeared normal by fluorescent visualization of Lucifer Yellow CH-loaded cells. It is concluded that cysteine cathepsins are the major or sole cytotoxic agents released from lysosomes by C12-Im. These observations also confirm the previous conclusions that C12-Im kills cells as a consequence of lysosomal disruption.

Ammonium Chloride↗
PubMed1987

Killing of Saccharomyces cerevisiae by the lysosomotropic detergent N-dodecylimidazole.

The lysosomotropic detergent N-dodecylimidazole (C12-Im) has previously been found to kill mammalian cells by concentrating in lysosomes, followed by lysosomal disruption and release of cytotoxic enzymes into the cytoplasm. The action of C12-Im on Saccharomyces cerevisiae is described in this report. C12-Im prevented growth of colonies when present in 1% yeast extract-2% Bacto-Peptone-2% glucose plates at concentrations of 5 micrograms/ml or above, or when present in a soft agar overlay at 20 micrograms/ml. Treatment of cells suspended in glucose-containing buffer (pH 8.0, 37 degrees C) with C12-Im (6 micrograms/ml) caused greater than 95% cell death within 6 min. Dependence of killing on C12-Im concentration was sigmoidal, suggesting a cooperative mode of action. Killing was pH dependent, being much more effective at pH 8.0 than at pH 5.0. Ammonium sulfate and imidazole protected against killing if added before, but not after, the addition of C12-Im. Sensitivity to C12-Im was strongly growth dependent: the cells were most sensitive at early to mid-logarithmic phase of growth and became progressively less sensitive during progression through late logarithmic and stationary phase. Vacuolar disruption by C12-Im was demonstrated by using cells loaded with lucifer yellow CH or fluoresceinated dextran in their vacuoles; vacuoles of logarithmically growing cells were more sensitive than those of stationary-phase cells. These results suggest that vacuolar disruption by C12-Im may underlie its cytotoxic effects.

Ammonium Sulfate↗
PubMed1987

Phenotypic revertants of temperature-sensitive M protein mutants of vesicular stomatitis virus: sequence analysis and functional characterization.

Twenty-five spontaneous temperature-stable revertants of four different temperature-sensitive (ts) M protein mutants (complementation group III: tsG31, tsG33, tsO23, and tsO89) were sequenced and tested for their ability to inhibit vesicular stomatitis virus RNA polymerase activity in vitro. Consensus sequences of the coding region of each M protein gene were determined, using total viral RNA as template. Fifteen different sequences were found among the 25 revertants; 14 differed from their ts parent by a single amino acid (one nucleotide), and 1 differed by two amino acids (two nucleotides). Amino acids were altered in various positions between residues 64 and 215, representing over 60% of the polypeptide chain. Resequencing of the Glasgow and Orsay wild types and the four ts mutants confirmed previously published differences (Y. Gopalakrishana and J. Lenard, J. Virol., 56:655-659, 1985), and one or two additional differences were found in each. The relative charges of the revertant M proteins, as determined by nonequilibrium pH gradient electrophoresis, were consistent with the deduced sequences in every case. The ability of each revertant M protein to inhibit the RNA polymerase activity of nucleocapsids prepared from its parent ts mutant was also tested. Only 13 of the 25 revertants had M protein with high (wild type-like) polymerase-inhibiting activity, while 5 had low (ts-like) activity, and 7 had intermediate activity, demonstrating that this property is not an essential concomitant of the temperature-stable phenotype. It is concluded that the high reversion frequency observed for these mutants arises from a very high incidence of pseudoreversion, i.e., many different molecular changes can repair the ts phenotype.

Amino Acid Sequence↗
PubMed1986

Radiation inactivation analysis of influenza virus reveals different target sizes for fusion, leakage, and neuraminidase activities.

The size of the functional units responsible for several activities carried out by the influenza virus envelope glycoproteins was determined by radiation inactivation analysis. Neuraminidase activity, which resides in the glycoprotein NA, was inactivated exponentially with an increasing radiation dose, yielding a target size of 94 +/- 5 kilodaltons (kDa), in reasonable agreement with that of the disulfide-bonded dimer (120 kDa). All the other activities studied are properties of the HA glycoprotein and were normalized to the known molecular weight of the neuraminidase dimer. Virus-induced fusion activity was measured by two phospholipid dilution assays: relief of energy transfer between N-(7-nitro-2,1,3-benzoxadiazol-4-yl)dipalmitoyl-L-alpha- phosphatidylethanolamine (N-NBD-PE) and N-(lissamine rhodamine B sulfonyl)-dioleoyl-L-alpha-phosphatidylethanolamine (N-Rh-PE) in target liposomes and relief of self-quenching of N-Rh-PE in target liposomes. Radiation inactivation of fusion activity proceeded exponentially with radiation dose, yielding normalized target sizes of 68 +/- 6 kDa by assay i and 70 +/- 4 kDa by assay ii. These values are close to the molecular weight of a single disulfide-bonded (HA1 + HA2) unit (75 kDa), the "monomer" of the HA trimer. A single monomer is thus inactivated by each radiation event, and each monomer (or some part of it) constitutes a minimal functional unit capable of mediating fusion. Virus-induced leakage of calcein from target liposomes and virus-induced leakage of hemoglobin from erythrocytes (hemolysis) both showed more complex inactivation behavior: a pronounced shoulder was present in both inactivation curves, followed by a steep drop in activity at higher radiation levels.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1985

Sequence alterations in temperature-sensitive M-protein mutants (complementation group III) of vesicular stomatitis virus.

Sequences were determined of the coding regions of the M-protein genes of the Glasgow and Orsay strains of vesicular stomatitis virus (Indiana serotype) and of two group III (M-protein) mutants derived from each wild type. Synthetic primers were annealed with viral genomic RNA and extended with reverse transcriptase. The resulting high-molecular-weight cDNA was sequenced directly. Both Glasgow and Orsay wild types differed in 13 bases from a clone of the San Juan strain sequenced by J. K. Rose and C. J. Gallione (J. Virol. 39:519-528, 1981). Six of these base changes caused amino acid changes in each wild type, whereas seven were degenerate. The Orsay and Glasgow sequences resembled each other more closely than either resembled that of Rose and Gallione, differing in eight nucleotides and four amino acids. Each of the four mutants, however, differed from its parent wild type in only one or two point mutations. Every mutation caused a change either from or to a charged amino acid; the change for tsG31 was Lys (position 215) to Glu, the change for tsO23 was Gly (position 21) to Glu, the change for tsO89 was Ala (position 133) to Asp, the changes for tsG33 were Lys (position 204) to Thr and Glu (position 214) to Lys. The charge differences predicted from these amino acid changes was confirmed by nonequilibrium pH gradient electrophoresis for tsG31, tsG33, tsO23, and the two wild types. These mutations affect residues spanning nearly 85% of the linear sequence, although the mutants possess nearly identical phenotypic properties.

Amino Acid Sequence↗
PubMed1984

Effects of DEAE-dextran on infection and hemolysis by VSV. Evidence that nonspecific electrostatic interactions mediate effective binding of VSV to cells.

The polycation DEAE-dextran increased the binding of VSV to BHK cells about fourfold over a wide range of VSV concentrations. The same proportion of bound virions was internalized by the cell in the presence or absence of DEAE-dextran. Viral primary RNA production was increased to the equivalent of a 4- to 4.5-fold increase in multiplicity of infection in the presence of DEAE-dextran, closely paralleling the increase in total VSV binding to the cell. Viral secondary RNA production was increased only to the equivalent of about twofold increase in multiplicity. The kinetics of both primary and secondary RNA production were indistinguishable in the presence or absence of DEAE-dextran. DEAE-dextran had to be present simultaneously with the input virions in order to enhance RNA production; addition even 30 min after infection was ineffective. Addition of the polycation DEAE-dextran was also required for appreciable VSV-induced hemolysis of human erythrocytes; erythrocytes of several other species were hemolyzed in the absence of DEAE-dextran, but hemolysis was enhanced by its presence. Maximal binding and hemolysis occurred at pH 5.0 and 37 degrees. Vesicles containing only G protein and viral lipid were 40% as hemolytic as intact virions at pH 5.0, but were inactive at pH 6.0; "spikeless" virions lacking G, or protein-free viral lipid vesicles were not hemolytic, showing that G protein is necessary for hemolysis. These results, together with other recent observations, suggest that multiple electrostatic interactions between VSV and the cell surface, rather than an affinity for specific surface molecules, mediated the productive (infection producing) binding of VSV to the cell surface.

Animals↗
PubMed1983

Interaction of enveloped viruses with planar bilayer membranes: observations on Sendai, influenza, vesicular stomatitis, and Semliki Forest viruses.

Exposure of a planar lipid bilayer to Sendai virus at pH 7.0 resulted in conductance increases that continued over several minutes, provided that the virus particles had first been conditioned by freezing and thawing, sonicating, or storing for 2 weeks in the cold. Individual electrical events could not be resolved, even on a millisecond time scale, and thus do not reflect the insertion of structural channels into the lipid bilayer. Prior treatment of the Sendai virions with protease prevented the conductance increases, but exposure of the bilayer to protease after induction of the conductance change did not abolish it. The Sendai-induced conductance change was increased in rate, but qualitatively unchanged in nature, if gangliosides were included in the planar bilayer. Activity for Sendai virus was low at pH 5.0, and increased with increasing pH up to 9.0. Influenza, Semliki Forest virus, and vesicular stomatitis virus all induced similar conductance changes around pH 5.2, but were inactive when tested at pH 7.0. The presence of cholesterol in the bilayer caused marked enhancement (two- to sixfold) of the response to Sendai, influenza and Semliki Forest virus, but caused only slight enhancement of the response to vesicular stomatitis virus. It is concluded that the observed increases in ionic permeability arise from alterations in lipid motions on a submillisecond time scale resulting from the incorporation of damaged viral membranes into the planar bilayer by fusion.

Cholesterol↗
PubMed1983

Cell killing by lysosomotropic detergents.

We have studied the mechanism by which lysosomotropic detergents kill baby hamster kidney cells. Lysosomotropic detergents are lysosomotropic amines (compounds with pK between 5 and 9, such as imidazole or morpholine) containing straight-chain hydrocarbon "tails" of 9-14 carbon atoms (Firestone, R. A., J. M. Pisano, and R. J. Bonney. 1979, J. Med. Chem., 22:1130-1133). Using lucifer yellow CH as a specific fluorescent label for lysosomes, it was shown by light microscopy that N-dodecyl (C12)-imidazole acted rapidly to damage lysosomes, causing leakage of dye into the cytoplasm. This was followed at later times by vacuolization, blebbing of the plasma membrane, cell rounding, and cell death. 3H-labeled C12-imidazole rapidly diffused into cells where much of it was trapped in lysosomes as shown by its co-migration with lysosomes in Percoll gradients. Cells preincubated with C12-imidazole released it slowly into C12-imidazole-free media, permitting the cells to be killed by the preincubation dose. Cell killing by the lysosomotropic detergents exhibited strongly sigmoidal dose-response curves. The sensitivity of baby hamster kidney cells to killing by C12-imidazole was density dependent, the cells being most sensitive at lowest cell densities, and relatively resistant at confluence. The amount of 3H-C12-imidazole taken up by the cells was also density dependent, with highest specific uptake occurring at the lowest cell density. A rise in lysosomal pH, measured in fluoresceinated dextran-labeled cells, commenced immediately upon addition of C12-imidazole to cells, and continued for over an hour. This was followed after a lag of 1-2 h by inhibition of protein and RNA synthesis and by lactate dehydrogenase release. Ionophores or lysosomotropic amines, such as methylamine, that raise intralysosomal pH provided substantial protection of the cells from killing by lysosomotropic detergents. These findings provide strong support for the idea that lysosomotropic detergents kill cells by disrupting lysosomes from within.

Animals↗
PubMed1982

A membrane glycoprotein that accumulates intracellularly: cellular processing of the large glycoprotein of LaCrosse virus.

The intracellular transport and certain posttranslational modifications of the large glycoprotein (G1) of LaCrosse virus (LAC) in BHK cells have been studied. G1 from released LAC virus was characterized by complex oligosaccharides (endo H-resistant) and covalently attached fatty acid. Only a small fraction of total cellular G1 was present on the baby hamster kidney cell surface. Cell-surface G1 contained complex oligosaccharides, while total G1 in infected cells contained largely unprocessed (endo H-sensitive) oligosaccharides. In addition, cell G1 contained significantly less fatty acid than virion-associated G1. Pulse-chase experiments showed that the oligosaccharides of G1 were processed to the complex from much more slowly than the oligosaccharides of the vesicular stomatitis virus (VSV) glycoprotein (G). In addition, transit of LAC G1 to the cell surface and into extracellular virions was two to three fold slower than the transit of VSV G. Thus LAC G1 accumulates intracellularly and is only slowly processed by intracellular processing enzymes. Treatment with monensin caused accumulation in the cell of a form of G1 with partial sensitivity toward endo H, suggesting that monensin may act to inhibit the glycosylation process directly.

Animals↗
PubMed1982

Ultraviolet-irradiated vesicular stomatitis virus and defective-interfering particles are similar non-specific inhibitors of virus infection.

The way in which ultraviolet-irradiated vesicular stomatitis virus (VSV) inhibits the early events in VSV infection has been further characterized. Comparison of several different u.v.-irradiated thermolabile, temperature-sensitive mutants before and after heat inactivation established a requirement for inhibitory activity of functional G, N and L proteins, but not M protein. Defective-interfering (DI) particles, whether irradiated or not, inhibited VSV primary transcription as efficiently as UV-VSV, suggesting that virus proteins rather than transcription products are responsible for inhibition. Addition of inhibitory UV-VSV at different times after infection established that inhibition results from an action at an intracellular site, rather than at the cell surface or in the process of internalization. A similar inhibition by UV-VSV of infection by Sendai virus, Semliki Forest virus, Sindbis virus and influenza virus suggests that UV-VSV is acting by inducing a general change in the intracellular environment.

Animals↗
PubMed1982

pH dependence of influenza A virus-induced haemolysis is determined by the haemagglutinin gene.

The pH-dependent profiles of haemolysis by influenza viruses AHK/8/68 (HK virus) and APR/8/34 (PR8 virus) were found to possess characteristic differences. Both viruses were highly haemolytic at pH 5.0, but while HK virus-induced haemolysis was undetectable above pH 5.5 to 5.6,, PR8 virus-induced haemolysis persisted at higher pH values, becoming undetectable above pH 5.7 to 5.8. In order to determine whether these haemolysis profiles were genetically determined, we studied the pH dependence of haemolysis by eight recombinants of HK and PR8 viruses. The pH profile of each recombinant clearly resembled that of one parent or the other, showing that it is an inherited trait. The pH profile was found to be conferred exclusively by the haemagglutinin (HA) gene.

Hemagglutinins, Viral↗
PubMed1981

Interactions of wild-type and mutant M protein of vesicular stomatitis virus with viral nucleocapsid and envelope in intact virions. Evidence from [125I]iodonaphthyl azide labeling and specific cross-linking.

Four different temperature-sensitive M protein mutants (tsM) of vesicular stomatitis virus (VSV) were characterized with regard to the association of the mutated M protein either with nucleocapsids or with membranes in the intact virions. Virions were labeled with the photoreactive hydrophobic probe [125I]iodonaphthyl azide (INA) to assess interactions between viral proteins and the lipid envelope. In wild type (wt) virions, the three major structural proteins--G, M, and N--were labeled in the ratio ca. 1.0:0.4:0.2. INA labeled only the membrane-associated peptide of G protein, both in the intact virion and in reconstituted G protein--viral lipid vesicles, demonstrating the specificity of INA for lipid bilayer regions. Labeling of tsM virions with INA resulted in a 2--3-fold greater incorporation into M protein than was found for wt virions, suggesting increased M--membrane associations in the mutant virions. Temperature-stable revertants from tsM possessed wt labeling characteristics. Interaction of the M protein with nucleocapsids was assessed from the abundance of disulfide-linked M--N complexes found after disruption of the virions by sodium dodecyl sulfate solution under nonreducing conditions. The abundance of such complexes was 30--80% less from tsM virions than from wt virions, suggesting decreased M--nucleocapsid interactions in tsM virions. Temperature-stable revertants from tsM resembled wt in the abundance of M--N complex formed. We conclude that the mutations alter M protein in such a way as simultaneously to increase its association with membrane and to decrease its affinity for nucleocapsids in the intact virion.

Animals↗