Biomedical subjects
Publications and source records attributed to J Lenard.
Hormones and hormone-binding proteins resembling those of vertebrates are widespread in fungi, yeast and bacteria. Functional responses of microbial cells to mammalian hormones have also been found. The evolutionary roots of the vertebrate endocrine system may, therefore, be far more ancient than is generally believed.
Multidrug resistance (MDR) in cultured cells and tumors is associated with overproduction of P-glycoprotein, a plasma membrane efflux pump normally present at very low levels. The cytotoxic action of N-dodecylimidazole (C12-Im), a lysosomotropic detergent, on cultured cells was previously shown to be strongly dependent on growth state, with rapidly growing cells being most sensitive and confluent cells most resistant. We show here that this may be due to a growth dependent increase in cellular P-glycoprotein activity. Both verapamil and nifedipine, structurally unrelated P-glycoprotein inhibitors, increased markedly the sensitivity of CHO fibroblasts to killing by C12-Im; the increase was greater in confluent than in growing cells. Also, verapamil inhibitable 3H-daunomycin efflux was more efficient from confluent than from subconfluent cells. The MDR cell line CH(R)C5 differed from all cell lines previously examined in that it did not show a growth-dependent decrease in C12-Im sensitivity, and sensitivity was not increased by verapamil or nifedipine. We suggest that a growth-dependent increase in MDR activity is a general property of cultured cells, except for those specifically overexpressing P-glycoprotein.
PDR4 is a gene that confers pleiotropic drug resistance (pdr) to the yeast Saccharomyces cerevisiae when present in high copy number [Leppert et al., Genetics 125 (1990) 13-20]. Transposon insertion mutations had identified the active region of the gene as a 3.7-kb SalI-EcoRI restriction fragment of the 8-kb cloned fragment. We have confirmed this by showing that this fragment is sufficient to confer pdr, and have sequenced its entire 3761 bp. It contains a single complete open reading frame (ORF) extending from nucleotide (nt) position 1631-3580, coding for a protein of 650 amino acids (aa). A 2.7-kb fragment containing this ORF is also sufficient to confer pdr. The aa sequence contains no recognizable homologies or consensus sequences, so it is a novel protein of unknown function. It is apparently soluble, since no transmembrane-type sequences were predicted. A second, partial ORF was also found, on the opposite strand, extending from nt position 774 to past the SalI site, which is apparently unrelated to pdr.
The protein that is responsible for specific, high-affinity binding of insulin to the surface of Neurospora crassa cells has been purified to homogeneity. The insulin binding activity of solubilized plasma membranes resembled that of intact cells with regard to affinity of binding, specificity for mammalian insulins, and amount of insulin bound per cell. Insulin binding activity was purified from Triton X-100 solubilized membranes in two steps: FPLC on a MonoQ HR5/5 column; and affinity chromatography on insulin-agarose. The pure material migrated as a single band of ca. 66 kDa on SDS gels, pI = 7.4 by isoelectric focusing. The protein bound 5.34 pmol of insulin/micrograms, or 35% of that expected for univalent binding. Cross-linking of 125I-insulin to pure protein or to solubilized membranes revealed a single labeled band of 67-70 kDa on SDS gels. In nonreducing native gels, two labeled bands of ca. 55 and 110 kDa were produced after cross-linking, and two bands of similar molecular weight bound iodinated insulin after transfer to nitrocellulose filters. These may correspond to active monomer and dimer forms. The pure protein possessed no protein kinase activity against itself, or against exogenous substrates (histone H2, casein, or the synthetic peptide Glu80-Tyr20), and possessed no detectable phosphorylated amino acids. It is suggested, however, that this 66-kDa protein is the "receptor" that mediates insulin-induced downstream metabolic effects.
A segment of DNA was amplified from the Neurospora crassa genome by the polymerase chain reaction using several oligonucleotides coding for highly conserved domains in proinsulin as primers and probe. A genomic clone corresponding to this segment was isolated and the nucleotide sequence was determined. The deduced amino acid sequence of a part of this segment bears remarkable resemblance to preproinsulin, but lacks several requirements for transcription or translation and must therefore be considered to be a pseudogene.
1) Insulin stimulated the phosphorylation of at least 14 discrete proteins in Neurospora crassa cells. Specific proteins were phosphorylated at serine, threonine, and tyrosine residues, as determined by phosphoamino acid analysis of discrete spots on two-dimensional gels. 2) Insulin stimulated the phosphorylation by [gamma-32P]ATP of at least six discrete proteins in solubilized N. crassa membrane preparations at serine and tyrosine residues. 3) A phosphotyrosine-containing protein of 38 kDa, pI 7.0-7.2, reacted by both immunoblotting and immunoprecipitation with antiserum to P2, a peptide from the human insulin receptor that contains an autophosphorylated tyrosine residue. In N. crassa cells, therefore, as in mammalian cells, insulin induces a variety of protein phosphorylations, some of which may be part of an evolutionarily conserved signal transduction pathway.
We have examined the fate of input viral proteins following the uncoating of vesicular stomatitis virus (VSV) by immunofluorescence microscopy, immunoelectron microscopy, and cell fractionation. VSV was adsorbed to BHK cells and allowed to become internalized in the presence of 100 mM NH4Cl; the NH4Cl was then removed to initiate synchronized uncoating. The three major structural proteins of VSV, the matrix protein (M), the nucleocapsid protein (N), and the glycoprotein (G), were each distributed uniquely after uncoating. Immunofluorescence microscopy showed that both G and N proteins retained a punctate distribution, whereas M protein was diffusely distributed throughout the cytoplasm, suggesting that it had become soluble. Immunoelectron microscopy showed that N protein was found in clusters (presumably in intact nucleocapsids) associated with the cell cytoskeleton and in unfused virions in endosomes and lysosomes. M protein was found diffusely distributed throughout the cytoplasm and also in endosomes and lysosomes. G protein was found only in association with endosomes and lysosomes after uncoating. Electrophoretic analysis of the high-speed cytosol fraction from infected cells showed that it contained chiefly M protein. The amount of M protein in the cytosol increased continuously during 90 min of uncoating, confirming its solubilization during uncoating. M protein was not covalently modified by phosphorylation upon uncoating, as evidenced by its mobility on nonequilibrium pH gradient gel electrophoresis. We suggest that those nucleocapsids associating with the cytoskeleton after uncoating may represent the sites of primary viral transcription.
23Na-NMR has been used to investigate some factors influencing the sodium content of a wall-less strains of Neurospora crassa. The shift reagent Tm(DOTP)H2(NH4)3 proved useful for this purpose, while several other reagents, previously used by others, were found to be unsuitable for use with these cells. When the cells were grown, washed and resuspended in medium containing sodium (25.3 mM), the intracellular sodium concentration was calculated to be 11.9 +/- 1.4 mM. This value rose within two minutes of addition of glucose (100 mM), to greater than 14 mM. Preincubation of cells with insulin (100 nM) had a significant effect on the subsequent rate of sodium accumulation during the period 3-12 minutes following glucose addition. Insulin-treated cells showed a slow, continued accumulation of sodium during this period (+1.14 +/- 0.39%/min), while control cells lost sodium very slowly (-0.63 +/- 0.29%/min; P of difference = 0.005).
The diadenylate triphosphates ppp5'A2'p5'A and ppp5'A3'p5'A were found to inhibit the purified RNA polymerase ('nucleocapsid') complex from vesicular stomatitis virus (VSV). The corresponding diadenylate monophosphate p5'A2'p5'A did not inhibit, nor did the triadenylate triphosphate ppp5'A2'p5'A2'p5'A; the diadenylate diphosphate pp5'A2'p5'A had intermediate inhibitory activity. Increasing the concentration of ATP, GTP or CTP in the reaction mixture decreased inhibition by ppp5'A2'p5'A, while UTP had minimal or no protective effect. ppp5'A2'p5'A did not protect the RNA polymerase from inactivation by N-ethylmaleimide. This suggests that the action of ppp5'A2'p5'A occurs at a site on the enzyme that is distinct from the N-ethylmaleimide-protecting, ATP-binding site characterized previously.
The relationship between NS protein phosphorylation and RNA polymerase activities was determined in nucleocapsids purified from vesicular stomatitis virus grown in BHK cells. Phosphate incorporation into endogenous NS protein under transcription conditions reached a maximum value of 0.06 mol/mol of NS within 20 to 30 min, while RNA synthesis remained linear for 90 min. Phosphate incorporation into NS increased further upon addition of kinase-free NS protein but not upon addition of nucleocapsid kinase (prepared as described below), indicating that cessation of NS phosphorylation under transcribing conditions was due to substrate exhaustion. When NS was phosphorylated with 32P, less than 8% of the radiolabel was lost during subsequent transcription, indicating that this phosphate did not turn over. Treatment of nucleocapsids with 5'-p-fluorosulfonylbenzoyl adenosine resulted in greater than 90% inhibition of NS phosphorylation but had no effect on RNA polymerase activity. Fast protein liquid (Superose-6) chromatography of a nucleocapsid (L + NS) fraction resulted in complete separation of the viral (L + NS) protein from NS-phosphorylating activity. The addition of this kinase-free (L + NS) fraction to a kinase-deficient N-RNA fraction reconstituted an active RNA polymerase containing less than 20% of the original NS-phosphorylating activity. These results demonstrate that NS-phosphorylating activity is unnecessary during vesicular stomatitis virus RNA synthesis and indicate that all of the protein kinase(s) present in purified nucleocapsids is probably of cellular rather than viral origin.
The membrane-reactive, photoactivatable probe 125I-TID [3-(trifluoromethyl)-3-(m-[125I]iodophenyl)-3H-diazirine] was found to label the M protein of vesicular stomatitis virus about 40% as much as G protein in intact virions, in agreement with labeling studies with other probes. By analyzing limited tryptic digestion and specific chemical cleavage products, the label was essentially entirely localized within the first 19, and probably within the first 5 to 10, amino acid residues at the N terminus, identifying this short amphipathic segment as the likely site of interaction of M protein with the viral bilayer.
The sensitivity of the human promyelocytic cell line HL60 to killing by the lysosomotropic detergent N-dodecyl imidazole (C12-Im) has been investigated in the exponential and stationary growth states and before and after differentiation induced by suitable effector molecules. Undifferentiated HL60 cells were more sensitive to killing by C12-Im in the rapid (exponential) phase of growth than in the stationary phase, in keeping with our observations on many other cell lines. Differentiation into granulocytes induced by dimethyl sulfoxide, or into macrophages induced by phorbol ester, resulted in a further dramatic decrease in sensitivity to C12-Im, as compared to undifferentiated HL60 cells in stationary phase. Viable cells remaining after treatment with C12-Im (60 micrograms/ml, 2 h) were: 0% for exponentially growing undifferentiated cells; 16% for stationary undifferentiated cells; 41% for differentiated granulocytes; and 29% for differentiated macrophages. Treatment with the cysteine cathepsin inhibitor L-trans-epoxysuccinylleucylamido(4-guanido)butane (E64) conferred resistance to C12-Im, showing that, in these cells, as previously demonstrated for Chinese hamster ovary fibroblasts, cysteine proteases were major cytotoxic agents involved in killing by C12-Im. Cell cathepsin B + L activity levels were dramatically reduced in those cells differentiated into granulocytes (11.2 units/mg of protein) and into macrophages (9.8 units/mg of protein) as compared with undifferentiated HL60 promyelocytes in stationary phase (30.4 units/mg of protein), correlating well with reduced sensitivity to C12-Im in the differentiated cells.
13C NMR and 31P NMR have been used to investigate the metabolism of glucose by a wall-less strain of Neurospora crassa (slime), grown in a supplemented nutritionally defined medium and harvested in the early stationary stage of growth. With D-[1-13C]- or D-[6-13C]glucose as substrates, the major metabolic products identified from 13C NMR spectra were [2-13C]ethanol, [3-13C]alanine, and C1- and C6-labeled trehalose. Several observations suggested the existence of a substantial hexose monophosphate (HMP) shunt: (i) a 70% greater yield of ethanol from C6- than from C1-labeled glucose; (ii) C1-labeled glucose yielded 19% C6-labeled trehalose, while C6-labeled glucose yielded only 4% C1-labeled trehalose; (iii) a substantial transfer of 13C from C2-labeled glucose to the C2-position of ethanol. 31P NMR spectra showed millimolar levels of intracellular inorganic phosphate (Pi), phosphodiesters, and diphosphates including sugar diphosphates and polyphosphate. Addition of glucose resulted in a decrease in cytoplasmic Pi and an increase in sugar monophosphates, which continued for at least 30 min. Phosphate resonances corresponding to metabolic intermediates of both the glycolytic and HMP pathways were identified in cell extracts. Addition of insulin (100 nM) with the glucose had the following effects relative to glucose alone: (i) a 24% increase (P less than 0.01) in the rate of ethanol production; (ii) a 38% increase (P less than 0.05) in the rate of alanine production; (iii) a 27% increase (P less than 0.05) in the rate of glucose disappearance. Insulin thus increases the rates of production of ethanol and alanine in these cells, in addition to increasing production of CO2 and glycogen, as previously shown.
Cells of the wall-less ("slime") strain of Neurospora crassa possess specific high affinity insulin binding sites on their cell surface. 125I-labeled bound insulin was not displaced from these cells by insulin-like growth factor II (IGF-II), and was only weakly displaced by IGF-I and proinsulin. Cross-linking of 125I-labeled insulin with N. crassa cells using disuccinimidyl suberate resulted in the labeling of a single band of ca. 67 kDa m.w. on a polyacrylamide gel. Two proteins of ca. 66 and 59 kDa m.w. were purified from detergent solubilized plasma membrane preparations by passage over an insulin-agarose affinity matrix. Antibodies against an autophosphorylation site on the human and Drosophila insulin receptors (anti P2) immunoprecipitated a single phosphoprotein of ca. 50 kDa m.w. from detergent solubilized plasma membranes, which possessed protein tyrosine kinase activity when histone H2 was used as substrate.
Sendai mutant ts271 contains less than 5% of the amount of HN glycoprotein found in wild-type Sendai. Fusion of this mutant with cardiolipin liposomes revealed no differences from the wild-type virus with regard to specific activity, pH dependence, or radiation inactivation. Target sizes of both mutant and wild-type viral proteins were determined by the radiation-induced disappearance of each band from an SDS-polyacrylamide gel and no differences were found. Of the viral proteins, only F had a target size corresponding to the monomer molecular weight, ca. 60 kDa, identical to the minimum unit previously determined by functional assay for Sendai virus-erythrocyte membrane fusion (K. Bundo-Morita, S. Gibson, and J. Lenard, Biochemistry 26, 6223-6227 (1987)). This provides additional evidence that F alone is the active protein mediating Sendai-erythrocyte fusion. It is concluded that the HN protein is unlikely to mediate any fusion reactions of the intact virions, either with biological membranes or with cardiolipin liposomes.
Radiation inactivation analysis was used to determine the size of the functional unit responsible for fusion of vesicular stomatitis virus (VSV) with cardiolipin or phosphatidylcholine-phosphatidylethanolamine (1:1) liposomes, and for VSV-induced hemolysis. When radiation-insensitive background values were subtracted, the calculated functional units for all three activities were similar, ranging from 866 to 957 kDa, equivalent to about 15 G protein molecules. This is in striking contrast to results of similar studies with influenza and Sendai viruses, in which the functional unit corresponded in size to a single fusion protein monomer, and suggests that VSV fusion may occur by a different mechanism.
Addition of mammalian insulin to a nutritionally rich, chemically defined culture medium affects Neurospora crassa "slime" (wall-less) cells, as indicated by enhancement of growth, extension of viability at the stationary phase of growth, alteration of morphology, and stimulation of glucose oxidation. Bovine, porcine, and recombinant human insulin had similar effects on growth and morphology, while proinsulin, reduced insulin, and several other proteins were inactive. Insulin added in the presence of excess antiinsulin antibody was without activity. Intact cells possessed high affinity insulin-binding sites, represented by a curvilinear Scatchard plot, suggesting that effects are mediated through insulin receptors on the cell surface. These findings establish a role for insulin or insulin-like molecules in regulating growth and metabolism in this fungal cell and demonstrate a close similarity to insulin effects on certain mammalian cells.
Addition of bovine insulin to cells of the wall-less variant FGSC4761 of Neurospora crassa ("slime") produced several significant effects on glycogen metabolism. 1) Intracellular levels of the glycogen precursor UDP-glucose decreased 17-18% (P less than 0.01) within 30 min of insulin addition. 2) Cells grown with insulin possessed 40% more glycogen than did control cells. 3) The incorporation of 14C-labeled glucose into glycogen increased 41% after 30-min treatment with 100 nM bovine insulin (P less than 0.01). 4) Insulin treatment of the cells caused activation of the enzyme glycogen synthase from a glucose-6-phosphate-dependent form to an independent form. Half-maximum activation occurred with 2 nM insulin. These are similar to insulin-induced effects in some mammalian cells. In contrast, no insulin-induced effect on glucose transport could be demonstrated in these cells.