Biomedical subjects

N L Davis

Publications and source records attributed to N L Davis.

At least 55 records · Page 3Linked to original sources

PubMed1991

Attenuating mutations in the E2 glycoprotein gene of Venezuelan equine encephalitis virus: construction of single and multiple mutants in a full-length cDNA clone.

Attenuated mutants of Venezuelan equine encephalitis virus (VEE) were isolated by selection for rapid penetration of cultured cells (R. E. Johnston and J. F. Smith, 1988, Virology 162, 437-443). Sequence analysis of these mutants identified candidate attenuating mutations at four loci in the VEE E2 glycoprotein gene: a double mutation at E2 codons 3 and 4, and single substitutions at E2 76, 120, and 209. Each candidate mutation was reproduced in an isogenic recombinant VEE strain using site-directed mutagenesis of a full-length cDNA clone of VEE. Characterization of these molecularly cloned mutant viruses showed that mutation at each of the four loci in the E2 gene was sufficient to confer both the accelerated penetration and attenuation phenotypes. Inoculation of the molecularly cloned viruses into rodent models that differ in their response to VEE suggested that individual mutations affected different aspects of VEE pathogenesis. Full-length clones containing multiple mutations were produced by combining independently attenuating mutations. Molecularly cloned viruses carrying two or three mutations were more attenuated in sensitive animal models than viruses which contained any single mutation alone. However, these highly attenuated strains still retained the ability to induce an immune response sufficient to protect against a high dose challenge with virulent VEE. These results indicate that production of a molecularly cloned live virus vaccine for VEE is feasible.

Animals↗

PubMed1990

Antigenic and genetic characterization of Sindbis virus monoclonal antibody escape mutants which define a pathogenesis domain on glycoprotein E2.

The Sindbis virus mutant SB-RL, in contrast to its parent, Sindbis strain AR339 (SB), is attenuated in neonatal mice, has an increased rate of penetration in tissue culture cells, and is more sensitive to neutralization by E2-specific monoclonal antibodies (MCAbs) R6 and R13. These phenotypic differences are controlled by substitution of an arginine for serine at amino acid 114 of the E2 glycoprotein. To explore these relationships further, MCAb R6 and R13 neutralization escape mutants of both SB and SB-RL were isolated and characterized. All mutants bound both MCAb R6 and R13 significantly less effectively in ELISA, and were more resistant to complement-mediated neutralization than their respective parental strains. Single coding changes in the E2 glycoprotein gene of each 11 mutants were identified. SB/R6, SB/R13, and SB-RL/R13 mutants contained a mutation at either E2 codon 96 or 159. SB-RL/R6 mutants contained changes at E2 codon 62, 96, or 159. These coding changes included two intragenic suppressor mutations. Mutation of E2 codon 159 from lysine to glutamate or codon 62 from asparagine to aspartate suppressed the attenuated phenotype conferred by E2 arginine 114 in SB-RL. However, only the change at E2 codon 62 significantly suppressed the rapid penetration phenotype of SB-RL. Mutation in E2 codon 96 of SB, replacing tyrosine with histidine, reduced the virulence of SB for neonatal mice but had no effect on penetration of cultured cells. Therefore, mutation in E2 codons 62, 96, 114, or 159 affected both virulence in animals and the binding or biological activity of these E2c-specific MCAbs. These results suggest that an E2 antigenic site (E2c), defined by MCAbs R6 and R13, is conformational in nature and may constitute a surface domain on Sindbis virions important for virulence in neonatal mice.

Animals↗

PubMed1989

Proton NMR study of the influence on iron oxidation/ligation/spin state on the heme orientational preference in myoglobin.

Proton nuclear magnetic resonance spectroscopy has been utilized to demonstrate that the degree of heme orientational disorder within a given myoglobin protein matrix can be a sensitive function of the oxidation/ligation/spin state of the heme iron. For sperm whale deuterohemin-reconstituted myoglobin, the equilibrium was found to strongly favor (5.7 to 7.8 kJ/mol) the X-ray characterized heme orientation in all six-coordinate states, but with a considerable reduction in preference (to 1.6 kJ/mol) in the five-coordinate deoxy state. In native yellow fin tuna myoglobin, changes in heme orientational preferences of approximately 3 kJ/mol occur even between two six-coordinate ferric states differing solely in spin states.

Animals↗

PubMed1989

In vitro synthesis of infectious venezuelan equine encephalitis virus RNA from a cDNA clone: analysis of a viable deletion mutant.

A molecular clone of Venezuelan equine encephalitis virus (VEE) was constructed from four cDNAs that were synthesized using the viral RNA genome as template. Together, these cDNAs are believed to represent all but the nine 5'-terminal nucleotides of the VEE genome sequence. A T7 promoter, followed by a single intervening G residue, and the exact 5'-terminus of VEE were added to the 5'-most clone using in vitro mutagenesis. Appropriate restriction fragments isolated from the cloned cDNAs were joined to form a candidate full-length VEE cDNA clone. RNA transcripts synthesized in vitro from the cDNA clone were able to initiate a productive infection in DEAE-dextran-treated chicken embryo fibroblasts (CEF). VEE antigens were demonstrated in RNA-transfected cells, and supernatants from transfected cultures contained infectious virus particles. The candidate full-length cDNA clone lacked 102 nucleotides of the VEE genome sequence. The deletion, which also was present in the genomes of progeny virions derived from the clone, did not appear to affect growth in cultured CEF, baby hamster kidney cells, or Vero cells. The site of the deletion was mapped to the 3'-end of the nsP3 gene by comparison to other alphavirus sequences. In this region, the VEE genome sequence includes two tandem 102-nucleotide repeats which can be arranged in a stable stem and loop structure. The sequence remaining in the deleted clone retains one copy of the duplicated sequence and, in addition, faithfully preserves a portion of the predicted stem.

Antigens, Viral↗

PubMed1988

Differences in virion stability among Sindbis virus pathogenesis mutants.

The structure of closely related Sindbis virus strains, which differ in their virulence for neonatal mice, has been probed by measuring the sensitivity of virions to heat, varying concentrations of dithiothreitol (DTT), phenylglyoxal or low pH. Attenuated mutants (SB-RL and SB-FP) were much more sensitive to loss of infectivity after heat or DTT treatment than either the prototype virulent strain (SB) or same-site virulent revertants of SB-RL. Incubation of SB-RL virions in the presence of DTT increased their density and exposed sulfhydryl groups on both E1 and E2 glycoproteins as determined by [14C]iodoacetamide labeling. Incubation of SB-RL at pH 6.1 for 1 h at 37 degrees C resulted in a 50% decrease in titer, whereas an equivalent decrease in SB titer was obtained at pH 6.0. Attenuated (SB-RL) and virulent (SB) strains could not be distinguished on the basis of phenylglyoxal sensitivity. Comparison of the nucleotide sequences of these strains and analysis of strains derived from recombinant full-length cDNA clones demonstrated that reduced virion stability under these conditions was a consequence of an arginine for serine substitution at position 114 of the surface glycoprotein E2. This same amino acid substitution is responsible for attenuation in neonatal mice. However, experiments with a second-site virulent revertant of SB-RL, which retained E2 arginine 114 and the reduced stability phenotype, indicated that virion instability in itself was not directly responsible for reduced virulence in vivo.

Animals↗

PubMed1988

Molecular analysis of Sindbis virus pathogenesis in neonatal mice by using virus recombinants constructed in vitro.

Genetic loci affecting Sindbis virus pathogenesis in neonatal mice have been examined by using a full-length cDNA clone of the virus (Toto1101). The full-length cDNA is linked to a bacteriophage SP6 promoter to facilitate the synthesis of infectious RNA transcripts in vitro. Virus derived from Toto1101 showed reduced virulence (attenuation) in neonatal mice. Replacement of the E1 glycoprotein and 6K genes of Toto1101 with cloned E1 and 6K genes derived from a virulent Sindbis virus strain, AR339 (SB), resulted in a new construct, TR2000, that gave rise to virulent virus. Sequence determinations for the entire substituted regions of TR2000, Toto1101, and related virulent and attenuated strains identified three coding differences in E1 between Toto1101 and TR2000. These differences, individually or in combination, may be responsible for the attenuated phenotype. Previous studies in this laboratory identified another attenuating mutation at amino acid position 114 of the E2 glycoprotein (N.L. Davis, F.J. Fuller, W.G. Dougherty, R.A. Olmsted, and R.E. Johnston, Proc. Natl. Acad. Sci. USA 83:6771-6775, 1986). Substitution of Arg-114 in the mutant SB-RL for Ser-114 of SB appears to confer three distinguishing phenotypes: attenuation in neonatal mice, increased sensitivity to specific E2 monoclonal antibodies, and accelerated penetration of BHK cells. Replacement of TR2000 sequences containing the codon for amino acid 114 of E2 with corresponding fragments from cDNA clones of SB or SB-RL produced two strains of Sindbis virus (TR2100 and TR2200) which were isogenic except for the E2 114 codon (Ser and Arg, respectively). The three diagnostic phenotypes cosegregated according to the origin of the codon for amino acid 114 of E2, confirming the dramatic effect of this single amino acid substitution on these three phenotypes.

Amino Acid Sequence↗

PubMed1988

Marketing and medicine. A basic guide for PAs.

Marketing is a conscious plan to influence the behavior of others. Physician assistants can use marketing techniques to promote themselves and their profession as well as to help build a practice and improve patient services. Basic components of a marketing strategy include analysis, planning, implementation, and monitoring. Marketing tools easily accessible to PAs are the telephone (call to check on compliance); a photocopy machine (patient handouts and practice newsletters); and the mail (postcard to remind of follow-up visit).

Marketing of Health Services↗

PubMed1987

Alternative forms of a strain-specific neutralizing antigenic site on the Sindbis virus E2 glycoprotein.

Experiments with monoclonal antibodies raised against two laboratory strains of Sindbis virus, SB and SIN, suggested the existence of a strain-specific neutralizing antigenic site (E2-b) on the E2 glycoprotein. A comparison of monoclonal antibody binding patterns and E2 glycoprotein gene sequences of six laboratory strains distinguished three different configurations of E2-b that correlated with specific amino acid substitutions at position 216 of the E2 glycoprotein. Further study of neutralization escape mutants selected with E2-b-specific antibodies confirmed that amino acid 216 is a major determinant of the E2-b antigenic site. Eight of nine mutants showed a coding change at position 216. One neutralization escape mutation created a new glycosylation site at position 213 and resulted in an E2 protein with an altered migration rate in SDS-PAGE. The neutralization escape mutants studied included amino acid substitutions not found in the laboratory strains that revealed differing binding requirements for two E2-b-specific monoclonal antibodies. The E2-b site is contrasted with the E2-c neutralizing antigenic site described previously (R.A. Olmsted, W.J. Meyer, and R.E. Johnston, 1986, Virology 148, 245-254).

Amino Acid Sequence↗

PubMed1986

A single nucleotide change in the E2 glycoprotein gene of Sindbis virus affects penetration rate in cell culture and virulence in neonatal mice.

The nucleotide sequence of the glycoprotein genes of fully virulent Sindbis virus and derived mutants that have reduced neurovirulence for neonatal mice (attenuated mutants) has been determined. A single amino acid difference, arginine instead of serine at position 114 of the mature E2 glycoprotein, distinguished the prototype attenuated mutant from its virulent wild-type parent. Virulent revertants of the attenuated mutant showed same-site reversion to the wild-type sequence. An identical single amino acid substitution, an arginine for the serine at E2 position 114, was found in a second independently selected attenuated mutant. The strains are characterized by genetic linkage between attenuation, accelerated penetration of baby hamster kidney cells, and efficient neutralization by the E2-specific monoclonal antibodies R6 and R13; selection for change in one property simultaneously selected for change in the other two (Olmsted, R. A., Baric, R. S., Sawyer, B. A. & Johnston, R. E. (1984) Science 225, 424-427 and Olmsted, R. A., Meyer, W. J. & Johnston, R. E. (1986) Virology 148, 1-10). The nucleotide sequence data suggest that a single mutation in the E2 gene is sufficient to cause these coordinate phenotypic changes. These findings identify a single locus in a Sindbis virus surface glycoprotein gene that determines both efficiency of interaction with cultured baby hamster kidney cells and degree of virulence in neonatal mice.

Amino Acid Sequence↗

PubMed1986

Hospital cost savings: resembling business.

Hospitals, like other employers, offer health care benefits to their employees. Cost savings can be realized when hospitals offer packages that resemble those provided by business employers. Business packages explain variance in cost savings more than employee characteristics do.

Age Factors↗

PubMed1986

Vesicular stomatitis virus N and NS proteins form multiple complexes.

The vesicular stomatitis virus nucleocapsid protein, N, associated specifically with the viral phosphoprotein, NS, in an in vitro system which supported vesicular stomatitis virus RNA replication. Essentially all the N protein was found complexed with NS. In addition, multiple forms of the N-NS complex were detected which differed in their sedimentation properties and ratios of N to NS.

Capsid↗

PubMed1985

Role of the nucleocapsid protein in regulating vesicular stomatitis virus RNA synthesis.

We describe experiments with two monoclonal antibodies to the vesicular stomatitis virus (VSV) nucleocapsid protein N with strikingly different characteristics. Antibody 1 binds to nucleocapsids and probably the pool of free (unbound) N protein; it inhibits transcription in vitro, and when microinjected into cells, protects the cells against VSV. Antibody 2 binds poorly to nucleocapsids, does not inhibit transcription, but when microinjected into cells, binds selectively to the free N and delays the appearance of progeny virus. We have confirmed these results by analyzing the effect of these antibodies on in vitro genomic RNA synthesis. The results of both the in vivo and in vitro experiments show that the replication of the VSV genome is controlled by the availability of the nucleocapsid protein, even when the polymerase has access to the host factors and multiple phosphorylated forms of the NS protein thought to be involved in genomic RNA synthesis.

Animals↗

PubMed1985

Nutritional requirements of Plasmodium falciparum in culture. I. Exogenously supplied dialyzable components necessary for continuous growth.

Continuous cultivation of Plasmodium falciparum presently requires the nutritionally complex medium, RPMI 1640. A basal medium of KCl, NaCl, Na2HPO4, Ca(NO3)2, MgSO4, glucose, reduced glutathione, HEPES buffer, hypoxanthine, phenol red (in RPMI 1640 concentrations), and 10% (v/v) exhaustively dialyzed pooled human serum was used to determine which vitamins and amino acids had to be exogenously supplied for continuous cultivation. Supplementation of basal medium with calcium pantothenate, cystine, glutamate, glutamine, isoleucine, methionine, proline, and tyrosine was necessary for continuous growth. This semi-defined minimal medium supported continuous growth of four isolates of P. falciparum at rates slightly less than those obtained with RPMI 1640. Adding any other vitamin or amino acid did not improve growth. Incorporation of several non-essential amino acids, particularly phenylalanine and leucine, into proteins was markedly enhanced in the minimal medium compared to RPMI 1640.

Amino Acids↗

PubMed1984

N protein alone satisfies the requirement for protein synthesis during RNA replication of vesicular stomatitis virus.

Genomic replication of the negative-strand RNA viruses is dependent upon protein synthesis. To examine the requirement for protein synthesis in replication, we developed an in vitro system that supports the genome replication of defective interfering particles of the negative-strand rhabdovirus vesicular stomatitis virus (VSV), as a function of protein synthesis (Wertz, J. Virol. 46:513-522, 1983). The system consists of defective interfering nucleocapsid templates and an mRNA-dependent reticulocyte lysate to support protein synthesis. We report here an analysis of the requirement for individual viral proteins in VSV replication. Viral mRNAs purified by hybridization to cDNA clones were used to direct the synthesis of individual proteins in the in vitro system. By this method, it was demonstrated that the synthesis of the VSV nucleocapsid protein, N, alone, resulted in the replication of genome-length RNA by both defective interfering intracellular nucleocapsids and virion-derived nucleocapsids. Neither the viral phosphoprotein, NS, nor the matrix protein, M, supported RNA replication. The amount of RNA replication for a given amount of N protein was the same in reactions in which either all of the VSV proteins or only N protein were synthesized. In addition, RNA replication products synthesized in reactions containing only newly made N protein assembled with the N protein to form nucleocapsids. These results demonstrate that the major nucleocapsid protein (N) can by itself fulfill the requirement for protein synthesis in RNA replication and allow complete replication, i.e., initiation and elongation, as well as encapsidation of genome-length progeny RNA.

Animals↗

PubMed1983

Heme orientational disorder in reconstituted and native sperm whale myoglobin. Proton nuclear magnetic resonance characterizations by heme methyl deuterium labeling in the Met-cyano protein.

The solution proton nuclear magnetic resonance spectrum of the Met-cyano form of sperm whale myoglobin reveals the presence of two sets of comparably intense resonances immediately after reacting the apoprotein with hemin, only one of which corresponds to that of the accepted native protein. Isotope labeling of individual methyl groups of hemin reveals that the methyl assignments differ characteristically in that similar resonance positions for the two components arise from the methyl groups related by a 180 degrees rotation about the alpha-gamma-meso axis. This phenomenon, observed earlier only for myoglobin with modified hemin, dictates that the second protein component in solution immediately after reconstitution must have the heme rotated by 180 degrees about the alpha-gamma-meso axis as compared to that found in the single crystal. The two components in the reconstituted protein equilibrate to yield the spectrum of the native Met-cyanomyoglobin for which there still exists approximately 8% of the minor component. Thus native myoglobin in solution is structurally heterogeneous in the heme pocket. Proton nuclear magnetic resonance spectra of deoxymyoglobin produced from both native and freshly reconstituted protein shown that the heterogeneity is also a property of the physiologically relevant reduced protein forms. It is suggested that, contrary to available X-ray data, heme orientational heterogeneity may be the rule rather than the exception in b-type hemoproteins, and that such disorder must be carefully considered in detailed correlations between structure and function even in native hemoproteins.

Animals↗

PubMed1983

Inhibition of vesicular stomatitis virus RNA synthesis by 2',3'-dideoxycytidine 5'-triphosphate.

The ability of the compound 2',3'-dideoxycytidine 5'-triphosphate (ddCTP) to serve as an inhibitor of viral RNA synthesis was examined using an in vitro system that supports vesicular stomatitis virus (VSV) protein synthesis, transcription and replication. Viral RNA synthesis was inhibited by 87 and 98% of control, respectively, in reactions containing 1 mM- and 10 mM-ddCTP in place of CTP. VSV RNA replication and transcription were inhibited equally by ddCTP. At a concentration of 1 mM-ddCTP, there was no inhibitory effect on viral protein synthesis; at 10 mM-ddCTP, total protein synthesis was inhibited by 30% as compared to control reactions. The presence of ddCTP had no effect on the size or relative molar amounts of each protein synthesized as analysed by electrophoresis on polyacrylamide gels. This is the first report describing a compound that will inhibit VSV RNA synthesis in vitro without compromising the concurrent synthesis and modification of proteins.

Deoxycytosine Nucleotides↗

PubMed1983

Cell-free synthesis and assembly of vesicular stomatitis virus nucleocapsids.

The association of newly synthesized vesicular stomatitis virus proteins into nucleocapsid structures was examined in a cell-free system that supports concurrent viral protein synthesis, transcription, and RNA replication. The vesicular stomatitis virus proteins synthesized by this system associated with the newly replicated RNA to form structures that banded in CsCl gradients with marker nucleocapsids. In reactions lacking nucleocapsid templates to program RNA synthesis, the newly synthesized proteins did not associate into nucleocapsid structures. The newly synthesized proteins associated with nucleocapsids were analyzed by electrophoresis on polyacrylamide gels containing sodium dodecyl sulfate after separation from non-associated proteins by chromatography on Bio-Gel A15M agarose columns. The results of this analysis showed that newly synthesized L, NS, and N proteins associated into nucleocapsids in the in vitro system. In addition, a small amount of newly synthesized M protein was stably bound to the nucleocapsids. The molar ratio of the associated, newly synthesized proteins was 2:350:1,000:10 (L:NS:N:M). More than 90% of the newly synthesized NS protein that associated with nucleocapsids in vitro was of the NS2 subspecies, as assayed by DEAE-cellulose column chromatography. The stability of the association of the newly synthesized proteins with nucleocapsids in the system mimicked that of the association of viral proteins with nucleocapsids from infected cells as measured by salt sensitivity. These data indicate that nucleocapsids were assembled from newly synthesized proteins within our in vitro system and that the molar ratio of assembled proteins was similar to that observed for virion nucleocapsids.

Capsid↗

PubMed1982

Synthesis of vesicular stomatitis virus negative-strand RNA in vitro: dependence on viral protein synthesis.

An in vitro system is described which supports the synthesis of vesicular stomatitis virus (VSV) negative-strand RNA. The major components of this system are (i) an mRNA-dependent rabbit reticulocyte lysate to carry out cell-free protein synthesis, (ii) the five VSV mRNAs to program VSV-specific protein synthesis, and (iii) nucleocapsids containing positive- and negative-strand genome-length RNA. The protein products synthesized in the system in response to addition of saturating amounts of the five VSV mRNA's included polypeptides which comigrated in acrylamide gels with the five VSV proteins. Approximately 200 pmol of protein per ml was synthesized during a 90-min reaction. The RNA products synthesized in the system included all five of the VSV mRNA's and, in addition, negative-strand, genome-sense RNA. All of the negative-strand RNA, which represented 2 to 5% of the total RNA product synthesized in vitro, banded in CsCl at the position of nucleocapsids. All of the mature mRNA's made in the system pelleted in CsCl. This technique allowed a clear separation of negative-strand product from the mRNA products and facilitated further analysis of the negative-strand product. The amount of negative-strand product produced in the system was shown to be a function of the amount of concurrent protein synthesis in the system. An increase in the level of protein synthesis led to an increase in the amount of negative-strand RNA synthesized, whereas inhibition of protein synthesis by cycloheximide resulted in a 70% inhibition of negative-strand synthesis. In contrast to the negative-strand RNA product, the amount of transcriptive product was decreased by 50% in the presence of maximum levels of viral protein synthesis. This inhibition was reversed by adding cycloheximide. Characterization of the negative-strand product by Northern blot analysis demonstrated that negative-strand product was being synthesized which hybridized to all five of the VSV mRNA's and, hence, that product representing all of the VSV cistrons was being made. This in vitro system offers an opportunity to study factors involved in the promotion of VSV genome replication as well as those responsible for the regulation of transcription.

Cell-Free System↗