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Biomedical subjects

L E Hightower

Publications and source records attributed to L E Hightower.

At least 55 records · Page 3Linked to original sources
PubMed1985

Induction of stress proteins in chicken embryo cells by low-level zinc contamination in amino acid-free media.

It has been reported that chicken embryo cells deprived of exogenous amino acids for 4 hours synthesize stress (heat-shock) proteins. Herein, we show that amino acid deprivation is not sufficient to cause induction of stress proteins. Zinc contaminating a component of commercial cell culture medium used to prepare amino acid-free medium was an inducer in our cultures. In the absence of exogenous amino acids, the concentration of zinc ions needed for half-maximal induction of stress proteins was an order of magnitude lower than the dose required for cells in complete medium. Histidine and cystine, which have high affinities for zinc ions, were the amino acids most effective in blocking the induction of stress proteins by zinc. Problems posed by heavy metal ions in culture media and biologic fluids for searches for in vivo inducers of the cellular stress (heat shock) response are discussed.

Amino Acids↗
PubMed1985

Differential induction of glucose-regulated and heat shock proteins: effects of pH and sulfhydryl-reducing agents on chicken embryo cells.

Glucose-regulated and heat shock proteins are two subsets of eukaryotic stress proteins that can be induced differentially, simultaneously, and reciprocally. Two new inducers, low extracellular pH and 2-mercaptoethanol, that stimulate chicken embryo cells to synthesize glucose-regulated proteins rapidly were found. Two classes of cellular targets for mercaptoethanol were defined operationally, one dependent on and the other independent of protein synthesis. A new inducer of heat shock proteins, high extracellular pH, was found as well. Inductions by low and high extracellular pH were inhibited by actinomycin D but were insensitive to cycloheximide. Inductions of glucose-regulated and heat shock proteins are discussed in terms of changes in intracellular pH and sulfhydryl oxidation states.

Animals↗
PubMed1984

Identification of a phosphorylated non-structural form of the P protein of Newcastle disease virus and analysis of P multimers.

Two phosphorylated and two non-phosphorylated variants of P protein isolated from Newcastle disease virions are known. Here, a fifth form of P was identified using two-dimensional polyacrylamide gel electrophoresis and peptide mapping. P form 5 was phosphorylated; however, unlike the four known variants of P, the new form was not a major protein in virions, which suggested an intracellular function. The subunit composition of four electrophoretically distinct, disulphide-linked multimers of P from virions was determined. Each homomultimer was composed of at least three molecules of a different one of the four virion-associated P variants.

Animals↗
PubMed1984

Stress mRNA metabolism in canavanine-treated chicken embryo cells.

Four major chicken stress mRNAs with apparent molecular weights of 1.2 X 10(6), 0.88 X 10(6), 0.59 X 10(6), and 0.25 X 10(6) to 0.28 X 10(6) were separated on acidic agarose-urea gels. Using cell-free translation, the coding assignments of these mRNAs were determined to be stress proteins with apparent molecular weights of 88,000, 71,000, 35,000, and 23,000. Despite high levels of translational activity in vivo and in vitro, no newly synthesized mRNA for the 23-kilodalton stress protein was detected on gels under conditions which readily allowed detection of other stress mRNAs, suggesting activation of a stored or incompletely processed mRNA. Cloned Drosophila heat shock genes were used to identify and measure changes in cellular levels of the two largest stress mRNAs. Synthesis of these mRNAs increased rapidly during the first hour of canavanine treatment and continued at a high rate for at least 7 h, with the mRNAs attaining new steady-state levels by ca. 3 h. Both of these inducible stress mRNAs had very short half-lives compared with other animal cell mRNAs. Using an approach-to-steady-state analysis, the half-lives were calculated to be 89 min for the mRNA encoding the 88-kilodalton stress protein and 46 min for the mRNA encoding the 71-kilodalton stress protein. Chicken 18S and 28S rRNA synthesis was inhibited, and actin mRNA levels measured with cloned cDNA encoding chicken beta-actin slowly declined in canavanine-treated cells.

Actins↗
PubMed1983

Biological consequences of neuraminidase deficiency in Newcastle disease virus.

A second-step revertant (L1) of a temperature-sensitive mutant (C1) of Newcastle disease virus agglutinated erythrocytes normally but had less than 3% of the wild-type (strain AV) levels of neuraminidase activity. Revertant L1 had seven times more virion-associated N-acetylneuraminic acid (NANA) than strain AV. NANA residues on purified virions were specifically labeled with periodate and tritiated borohydride. Analyses of radiolabeled L1 virions on sodium dodecyl sulfate-polyacrylamide gels showed that most of the virion-associated NANA was in a high-molecular-weight component with an electrophoretic mobility different from that of any known viral protein. NANA was also detected in molecules with the electrophoretic mobility of the viral glycoproteins HN and F1. Revertant L1 had a twofold lower rate constant of attachment to HeLa cells than that of the wild-type. Treatment of L1 virions with Vibrio cholerae neuraminidase removed the excess NANA and returned L1 attachment kinetics to normal. Revertant N1, which has 10-fold more neuraminidase activity than L1, penetrated host cells at the same rate as L1. L1 was impaired in elution from erythrocytes. Removal of virion-associated NANA exacerbated this defect. Despite a small disadvantage in attachment and a major defect in elution relative to strain AV, revertant L1 enjoyed a slight advantage over the wild-type during a single reproductive cycle in cultured chicken embryo cells.

Animals↗
PubMed1982

Maturation of the envelope glycoproteins of Newcastle disease virus on cellular membranes.

Based on subcellular fractionation data, the following maturation pathways were proposed for the Newcastle disease virus glycoproteins. During or shortly after synthesis in rough endoplasmic reticulum, hemagglutinin-neuraminidase (HN) and fusion (F0) glycoproteins underwent dolichol pyrophosphate-mediated glycosylation, and HN assumed a partially trypsin-resistant conformation. HN began to associate into disulfide-linked dimers in rough endoplasmic reticulum, and at least one of its oligosaccharide side chains was processed to a complex form en route to the cell surface. During migration in intracellular membranes, F0 was proteolytically cleaved to F1.2. Neither HN nor F1,2 required oligosaccharide side chains for migration to plasma membranes, and cleavage of F0 also occurred without glycosylation. Virion- and plasma membrane-associated HN contained both complex and high-mannose oligosaccharide chains on the same molecule, and F1,2 contained at least high-mannose forms. Several of the properties of HN were notable for a viral glycoprotein. The oligosaccharide side chains of HN were modified very slowly in chick cells, whereas those of the G glycoprotein of vesicular stomatitis virus were rapidly processed to a complex form. Therefore, their different rates of migration and carbohydrate processing were intrinsic properties of these glycoproteins. Consistent with its slow maturation, the HN glycopolypeptide accumulated to high levels in intracellular membranes as well as in plasma membranes. Intracellular HN contained immature oligosaccharide side chains, suggesting that it accumulated in the pre-Golgi/Golgi segment of the maturation pathway. The major site of accumulation of mature HN with neuraminidase activity was the plasma membrane.

Animals↗
PubMed1982

Revertant analysis of a temperature-sensitive mutant of Newcastle disease virus with defective glycoproteins: implication of the fusion glycoprotein in cell killing and isolation of a neuraminidase-deficient hemagglutinating virus.

Biological and molecular properties of a temperature-sensitive mutant (C1) of Newcastle disease virus and its revertants were analyzed. C1 exhibited three temperature-sensitive alterations (plaque formation, virion assembly, and cytopathogenicity) and several defects which were also present at the permissive temperature. C1 virions contained low amounts of hemagglutinin-neuraminidase glycopeptides and consequently were deficient in hemagglutinating and neuraminidase activities. These virions also contained defective fusion glycoproteins which rendered them poorly hemolytic and slow to penetrate cultured chicken embryo cells. The biological activities of the membrane glycoproteins were recovered sequentially in a series of plaque-forming revertants. The coreversion of hemolysis, membrane-penetrating activities, and cytopathogenicity in the first-step revertant (S1) suggested that fusion glycoproteins were major contributors to cellular destruction. This revertant also provided evidence of a role for fusion glycoproteins in virion assembly. From S1 we isolated a large-plaque-forming revertant (L1) that assembled wild-type amounts of biologically active hemagglutinin-neuraminidase glycoproteins into virions. Although it was normal for hemagglutination, L1 had less than 3% of the neuraminidase activity of the wild type, demonstrating that these two activities can be uncoupled genetically. The neuraminidase deficiency of L1 did not impair its virulence in ovo or its reproduction in cultured cells.

Animals↗
PubMed1982

Coding assignments of the five smaller mRNAs of Newcastle disease virus.

The polypeptide coding assignments for the five messengers of the 18S size class of Newcastle disease virus (NDV) RNA have been determined by cell-free translation of individual RNAs separated by gel electrophoresis. Listed in order of their decreasing electrophoretic mobilities in acid agarose-urea gels, the coding assignments of the RNAs were as follows: RNA 1, M protein; RNA 2, P protein; RNA 3, NP; RNA 4, F glycoprotein; and RNA 5, HN glycoprotein. RNA 2 also directed the synthesis of 33- and 36-kilodalton proteins, which were tentatively identified as being overlapping segments of the P protein. The 33- and 36-kilodalton polypeptides could be detected in infected cells, but not in purified virions of NDV. Since the other unique NDV RNA, a 35S species, has been shown previously to encode the viral L protein, these results complete the coding assignments of the six known NDV mRNAs.

Animals↗
PubMed1982

Newcastle disease virus stimulates the cellular accumulation of stress (heat shock) mRNAs and proteins.

A biological agent, Newcastle disease virus, stimulated the synthesis of stress proteins in cultured chicken embryo cells. Previously, only physical and chemical agents were known to induce these proteins. The levels of translatable stress mRNAs were elevated in cells infected with avirulent or virulent strains; however, stress protein synthesis was stimulated strongly only in cells infected by avirulent strains. As did several other paramyxoviruses, avirulent strains of Newcastle disease virus stimulated the synthesis of glucose-regulated proteins as well as stress proteins. Possible stimuli of the synthesis of these two sets of proteins in paramyxovirus-infected cells are considered.

Animals↗
PubMed1981

Cellular responses to stress: comparison of a family of 71--73-kilodalton proteins rapidly synthesized in rat tissue slices and canavanine-treated cells in culture.

Cultured rat embryo cells exposed to the L-arginine analogue L-canavanine rapidly accumulated a major 71 kilodalton polypeptide and several minor ones (110, 95, 88, and 78 kilodaltons). Canavanine-treated cultures contained elevated levels of translatable mRNA encoding P71, and the stimulated synthesis of this protein was blocked by actinomycin D, suggesting that P71 is inducible. Rat embryo cells maintained under routine culture conditions synthesized only trace amounts of P71; however, they accumulated an abundant 73 kilodalton protein that was closely related to P71. No kinetic evidence of a precursor-product relationship between P73 and P71 was found. The peptide map of P71 from cultured cells was identical to the map of proteins with the same electrophoretic mobility isolated from incubated slices of rat telencephalon. Previous studies (White, '80a, b, c) have shown that the latter proteins are rapidly synthesized by cells associated with cerebral microvessels in incubated brain slices, but are not detectable in vivo. Herein we present evidence that the synthesis of P71 is not unique to brain slices. Incubated slices of heart, lung, thymus, kidney, spleen, and liver all accumulated an abundant 71 kilodalton size class. The peptide maps of P71 obtained from brain, heart, lung and thymus tissue were similar. The stimulated synthesis of P71 in brain, heart, and lung slices was inhibited strongly by the addition of actinomycin D at the start of incubation. The 71-73 kilodalton proteins of canavanine-treated rat embryo cells and incubated slices from seven different organs were compared in detail on two-dimensional polyacrylamide gels. Eight charge variants were detected in extracts of lung, spleen, and thymus tissue, four in liver and heart, three in kidney, and two different pairs of variants in extracts of brain tissue and cultured cells. The possible significance of the rapid synthesis of a similar small set of proteins in tissue slices and cultured cells in response to a variety of physical, chemical, and biological stimuli is discussed in terms of cellular responses to traumatic injury and metabolic stress.

Animals↗
PubMed1981

Identification of the P proteins and other disulfide-linked and phosphorylated proteins of Newcastle disease virus.

A unique abundant protein, designated P by analogy to the putative polymerase proteins of other paramyxoviruses, was identified in purified Newcastle disease virus. Under nonreducing conditions the P proteins could be separated from other viral proteins on sodium dodecyl sulfate-polyacrylamide gels. The P proteins were isolated from detergent-solubilized virions as 53,000- to 55,000-dalton monomers and disulfide-linked trimers. Distinct forms of P having four different isoelectric points and two different electrophoretic mobilities were resolved by two-dimensional electrophoresis. Two forms of P were phosphorylated, as were the nucleocapsid protein and non-glycosylated membrane protein. In addition to disulfide-linked forms of P, dimers of the hemagglutinin-neuraminidase glycoprotein and two disulfide-linked versions of the fusion glycoprotein were identified. Several electrophoretic variants of the nucleocapsid protein that were probably created by intrachain disulfide bonding were also isolated from virions under nonreducing conditions. The locations of the newly identified proteins were determined by detergent-salt fractionation of virions and by surface-selective radioiodination of the viral envelope. The P proteins were associated with nucleocapsids and were not detected at the surface of virions. Both forms of the fusion glycoproteins were on the exterior of the viral envelope. Herein the properties of the P proteins are compared with similar proteins of rhabdoviruses and other paramyxoviruses, and a role for multiple forms of proteins in the genetic economy of newcastle disease virus is discussed.

Disulfides↗
PubMed1980

Cultured animal cells exposed to amino acid analogues or puromycin rapidly synthesize several polypeptides.

Four major acidic polypeptides, with molecular weights of 88, 72, 71, and 23 thousand, and minor polypeptides with molecular weights of 110, 50, 38, and 30 thousand rapidly accumulated in cultured chick embryo (CE) cells which were exposed for three hours to the arginine analogue canavanine. P110, P88, P71,72, and P23 had unique peptide maps. Evidence of a 27,000 dalton precursor to P23 was obtained. The analogue-stimulated proteins were not related to another set of inducible avian polypeptides known as the glucose-regulated proteins. In mammalian cells, the rate of accumulation of several polypeptides, which were similar in size to the avian proteins, sharply increased after canavanine treatment. Proteins with the same electrophoretic mobilities, isoelectric points, and peptide maps as the analogue-stimulated proteins were expressed at low levels in untreated cultures. To determine the time courses of the canavanine-mediated increases in protein accumulation and the recovery of protein metabolism after analogue treatment, radioactively labeled proteins were extracted from CE cells and analyzed on SDS-polyacrylamide gels. In cultures exposed to canavanine, the rates of accumulation of P88 and P71,72 increased from basal to new plateau levels in about 1.5 hours, while P23 required about 2.5 hours. When added with the analogue, actinomycin D and cordycepin blocked the increases in protein accumulation. These inhibitors also blocked the rapid decline in the rates of accumulation of the enhanced proteins which occurred after removal of canavanine. Studies of the matabolic stability of the enhanced proteins indicated that the changes in their accumulation were caused by alterations in their rates of synthesis. Thus, the analogue-mediated response fulfilled several of the criteria for inducible eucaryotic gene expression. The amino acid analogue p-fluorophenylalanine and the chain-terminating analogue of amino acyl-tRNA puromycin stimulated the synthesis of the same set of proteins induced by canavanine. The enhanced synthesis of these proteins appeared to be a cellular response to either the presence or catabolism of abnormal proteins and puromycyl peptides.

Amino Acids↗
PubMed1980

Transcriptional map for Newcastle disease virus.

A transcriptional map of Newcastle disease virus was determined by measuring the kinetics of UV inactivation of the transcription of individual genes and of viral infectivity. The inactivation of single genes was monitored by measuring the reduction in the accumulation of viral gene products in vivo and in vitro. In vivo, the accumulation of viral polypeptides in infected cells was measured after reversal of a cycloheximide treatment designed to inhibit secondary transcription. Actinomycin D and a hypertonic medium were used to decrease selectively the synthesis of host cell polypeptides in infected cells. In vitro, mRNA's synthesized by irradiated viruses were analyzed by translation in cell-free systems under conditions in which the amount of each polypeptide synthesized reflected the relative abundance of the corresponding mRNA. UV target sizes were obtained for the genes coding for the HN, F0, NP, M, L, and P polypeptides; the 47,000-dalton protein was not detected. A comparison of the UV target sizes with the corresponding gene sizes suggested that transcription of these genes initiated at a single promotor and proceeded in the order NP, P, (F0, M), HN, L. These experiments were performed with Newcastle disease virus strains Australia-Victoria and B1-Hitchner; for both strains, two forms of the P polypeptide which differed in electrophoretic mobility were detected. Proof that the P protein is virus specific was obtained. In addition, infection of chicken embryo cells with avirulent strain B1-Hitchner enhanced the accumulation of at least four polypeptides that appeared to be specified by the host cell rather than by the infecting virus.

Genes, Viral↗
PubMed1980

Maturation of the hemagglutinin-neuraminidase and fusion glycoproteins of two biologically distinct strains of Newcastle disease virus.

We have compared the glycoproteins of two biologically distinct virulent strains of Newcastle disease virus. Cells infected by either strain AV or HP produce infections virions and the cellular surfaces have hemadsorbing activity; however, only cells infected by strain AV undergo fusion from within. We fractionated chicken embryo cells and monitored the incorporation of radioactive proteins into virions to study the sites of synthesis, cellular locations, and kinetics of virion assembly for the hemagglutinin-neuraminidase (HN) and the fusion glycoproteins of each strain. We found that the HN glycoprotein of both strains was synthesized on rough endoplasmic reticulum (ER), accumulated in low-density membranes derived from smooth ER and the plasma membrane, and appeared in virions after a 30-min delay. The fusion glycoprotein of both strains was synthesized as a precursor in rough ER and subsequently processed to the active form. However, the sites of accumulation of the fusion glycoproteins were strain-dependent. The larger subunit F1 of the fusion glycoprotein of strain AV was detected in subcellular fractions enriched for plasma membranes, while that of strain HP accumulated in denser fractions which contained internal membranes. This result suggests that differential compartmentation of viral glycoproteins may influence the expression of biologic activities such as fusion on cellular surfaces. Despite their different sites of accumulation, the F1 glycoproteins or F-related polypeptides of similar size were assembled into virions of both strains. The kinetics of incorporation of this protein into virions appear to be too rapid for migration through internal membrane systems. Furthermore, tunicamycin did not block the incorporation of F-related polypeptides into virions. A hypothesis is presented to explain the unusual behavior of the F-related proteins.

Animals↗
PubMed1978

Transcription and translation of Newcastle disease virus mRNA's in vitro.

Transcription directed in vitro by Triton-activated Newcastle disease virus (NDV) was stimulated and prolonged by the presence of cytoplasmic extracts of animal cells. The RNA products closely resembled those of NDV transcription in vivo by several criteria: binding to oligodeoxythymidylic acid-cellulose, the mobility and relative abundance of each major band resolved by polyacrylamide gel electrophoresis, and the ability to direct the accurate cell-free synthesis of polypeptides corresponding to the NDV proteins HN, F0/F1, NP, and M. Synthesis of a novel polypeptide related to NP but of higher apparent molecular weight was also detected. These results indicated that cell-free transcription under these conditions was a close facsimile of NDV transcription in vivo. In addition, both in vitro and in vivo, NDV polypeptides were synthesized in nonequimolar amounts which reflected the order of the genes in the transcriptional map: NP, F0, M, (47K, HN), L. Strains AV and HP, virulent strains which have differences in biological activities, exhibited differences in the polypeptides synthesized in infected cells and in cell-free systems.

Cell-Free System↗
PubMed1975

Protein metabolism during the steady state of Newcastle disease virus infection. I. Kinetics of amino acid and protein accumulation.

Pulse-chase experiments and studies on the effects of varying pulse lenghts on radioactive amino acid and protein accumulation have been carried out to evaluate several possible mechanisms for the inhibition in cellular protein accumulation during infection of chicken embryo cells by Newcastle disease virus. The inhibition is probably at the level of synthesis of cellular protein since no evidence for either increased degradation of protein or alterations in cellular permeability to protein was found in infected cultures. The magnitude of the reduction in the rate of cellular protein accumulation and consequently total protein accumulation depend upon the length of the radioisotopic labeling period. In contrast, the rate of viral protein accumulation is independent of the length of the labeling period. A double-label difference analysis of polyacrylamide gels was used in all of the kinetics studies to distinguish between viral and cellular protein accumulation. An unstable fraction which could be labeled with radioactive amino acids was detected in both infected and uninfected cultures. This material migrated mainly in the 50,000- to 60,000-dalton region of polyacrylamide gels, exhibited saturation kinetics during accumulation studies, and turned over rapidly during a chase. The relative amount of this fraction was not affected by infection. Gel analysis of the radioactive protein recovered from the medium from both infected and uninfected cultures revealed a major component with an apparent molecular weight of 33,000. None of the major viral polypeptides could be detected in the medium after a 30-min chase following a brief labeling period.

Amino Acids↗
PubMed1975

Relationships among the polypeptides of Newcastle disease virus.

We have studied the relationships among the polypeptides of Newcastle disease virus by using both kinetic and tryptic peptide analyses. The results of our tryptic peptide analyses suggest that there are at least six unique viral polypeptides--L, HN, FO(F), NP, M, and a 47,000-dalton polypeptide. The small virion glycopolypeptide F is related to FO, a glycopolypeptide found only in infected cells. In addition, several smaller polypeptides, including a 53,000-dalton polypeptide found both in purified virions and in infected cells, are related to the nucleocaspid protein. Kinetic analysis of each viral polypeptide reveals that all of the major viral polypeptides, with the possible exception of L, are stable after an amino acid chase. A precursor-product relationship between FO and F was not demonstrable by pulse-chase experiments. Also, almost the same relative amount of F, the putative product, was present in infected cultures after either 5 or 30 min of radioisotopic labeling. These results suggest that FO is processed rapidly.

Glycopeptides↗