Search PubMed⌕ Search

Biomedical subjects

J F Young

Publications and source records attributed to J F Young.

At least 109 records · Page 6Linked to original sources
PubMed1983

Efficient expression of influenza virus NS1 nonstructural proteins in Escherichia coli.

RNA segment 8 of the influenza A virus genome codes for two nonstructural proteins, NS1 and NS2, for which the functions are unknown. Cloned cDNA copies of this gene from three different influenza A virus strains were inserted into an Escherichia coli plasmid expression vector, pAS1, carrying the strong regulatable lambda phage promoter, PL. After induction, the NS1 proteins were overproduced to levels of 20-25% of total cellular protein. This was surprising in that the codon composition for these eukaryotic genes is similar to that for weakly expressed proteins in E. coli. Thus, under the appropriate conditions, it appears that high level expression of genes containing a relatively large proportion of minor codons can be obtained. The NS1 protein produced in bacteria from a cloned cDNA copy of the A/PR/8/34 virus NS gene was purified to apparent homogeneity and used to generate a high-titer monospecific rabbit antiserum. Immunoprecipitation studies showed this antibody to be crossreactive against the NS1 proteins produced by several different influenza A virus strains. Immunofluorescence experiments in Madin-Darby canine kidney cells showed the NS1 proteins to be located in the nucleoplasm early in infection for all strains examined. With some of the strains, NS1-specific immunofluorescence was observed predominantly in the nucleoli later in infection. This technology can be used to obtain other viral proteins in pure form for structural, functional, and immunological studies.

Animals↗
PubMed1983

Sequential mutations in the NS genes of influenza virus field strains.

The complete nucleotide sequences of the NS genes from three human influenza viruses, A/FM/1/47 (H1N1), A/FW/1/50 (H1N1), and A/USSR/90/77 (H1N1), were determined. Only five single-base differences were found within the sequences of the A/FW/1/50 and A/USSR/90/77 NS genes, thus confirming earlier data suggesting that the 1977 H1N1 viruses are closely related to virus strains that were circulating around 1950. Comparison of all three sequences with those from A/PR/8/34 and A/Udorn/72 viruses illustrates that these genes (with the exception of that of the A/USSR/90/77 strain) evolve through cumulative base changes along a single common lineage. A nucleotide sequence variation of approximately 2.2 to 3.4% per 10 years was determined for the NS gene segments. Extensive size variation was also observed among the NS1 proteins of the various human viruses. The A/FM/1/47 NS1 protein, which consists of 202 amino acids, is 15% shorter than the A/Udorn/72 NS1 protein, which consists of 237 amino acids.

Amino Acid Sequence↗
PubMed1982

Variation of influenza A, B, and C viruses.

Influenza is caused by highly variable RNA viruses belonging to the orthomyxovirus group. These viruses are capable of constantly changing the genes coding for their surface proteins as well as for their nonsurface proteins. The mechanisms responsible for these changes in type A influenza viruses include recombination (reassortment) of genes among strains, deletions and insertions in genes, and, frequently, point mutations. In addition, old strains may reappear in the population. Influenza viruses of types B and C appear to vary to a lesser degree. The mechanisms responsible for changes in these viruses are not well characterized.

Genetic Variation↗
PubMed1982

Oligonucleotide mapping: evaluation of its sensitivity by computer-simulation.

A frequently used method of comparing large RNA molecules employs the two-dimensional display of oligonucleotides generated through the action of specific RNases (oligonucleotide mapping, fingerprinting). Using computer simulations and simple analytic expressions the number of large RNase T1-resistant oligonucleotides obtained from random RNA sequences can be estimated. The computer simulations also permit estimation of the number of large oligonucleotides which remain unchanged as random variations are introduced into a random RNA sequence. In addition, computer analysis also provides a means of estimating statistical confidence limits to be used in a quantitative comparison of fingerprints of different RNA molecules. The model shows that two RNA sequences which differ overall by 1%, 5% or 10% share, on average, only 85%, 50% or 25%, respectively, of their large oligonucleotides. Thus, the use of fingerprint analysis is recommended only when closely related RNAs or regions of RNAs are compared (sequence homology greater than 90%).

Base Sequence↗
PubMed1982

Caffeine disposition after oral doses.

Caffeine (TMX) disposition was studied in mean after 1, 5, and 10 mg/kg in water, as mocha coffee (1.54 mg/kg) and as a soft drink (0.22 mg/kg). TMX and its metabolites were analyzed in plasma and urine by high-pressure liquid chromatography. The design permitted confirmation of most of the partial results in various experimental settings and contributed new data on the metabolic disposition of TMX, with specific reference to main dimethylxanthine metabolite found in plasma, paraxanthine (1,7-dimethylxanthine). Different analysis methods were compared for the calculated parameters (absorption and elimination rate constants and renal clearance)to assess the consistency of results. The kinetics of TMX and of its dimethylated metabolites in plasma were described with a model that used an analogdigital hybrid computing system. In addition to providing a comprehensive profile of TMS disposition in the healthy adult, the results indicate tha TMX exhibits dose-independent kinetics at the levels at which man normally takes TMX.

Administration, Oral↗
PubMed1982

Evolution of influenza A and B viruses: conservation of structural features in the hemagglutinin genes.

The complete nucleotide sequence of the hemagglutinin (HA) gene of a type B influenza virus (B/Lee/40) was obtained by using cloned cDNA derived from the RNA segment. The gene is 1,882 nucleotides long and can code for a protein precursor of 584 amino acids. Structural features common to type A virus HAs are also conserved in the B virus HA. These include a hydrophobic signal peptide, hydrophobic NH2 and COOH termini of the HA2 subunit, and a HA1/HA2 cleavage site involving an arginine residue. The sequence of the B HA gene and its deduced amino acid sequence were compared to those of a type A influenza virus (A/PR/8/34). When these two genes were aligned, it was found that 24% of the amino acids in the HA1 subunits and 39% of the amino acids in the HA2 subunits are conserved. This degree of relatedness between type B virus and type A virus HAs (intertypic comparison) is similar to the homologies observed among certain type A virus HAs (intratypic comparison). A close evolutionary relationship is therefore suggested between the HAs of type A and type B influenza viruses.

Amino Acid Sequence↗
PubMed1981

Atmospheric pressure ionization mass spectrometry: the routine determination of 2,4,5-trichlorophenoxyacetic acid and its metabolites.

This study demonstrated the utility of negative ion atmospheric pressure ionization mass spectrometry for the routine quantitation of 2,4,5-trichlorophenoxyacetic acid and its glycine and taurine amide metabolites in mouse blood, urine and feces samples. The quantitation of 2,4,5-trichlorophenoxyacetic acid in blood follows a short cleanup procedure and used 2,4,5-trichlorophenoxy-13C6-acetic acid as the stable label isotope diluent. A more extensive cleanup procedure utilizing high-pressure liquid chromatography was required for the determination of 2,4,5-trichlorophenoxyacetic acid and its two metabolites in urine and feces. The glycine amide metabolite was quantitated by the 13C stable isotope diluent method. The taurine amide relied on an initial separation step followed by using a second 2,4,5-trichlorophenoxy-13C6-acetic acid spike fot its isotope diluent. Alkaline hydrolysis of the metabolites, followed by methylation, allowed the methyl ester of 2,4,5-trichlorophenoxyacetic acid to be solely used as the analyte in the negative ion atmospheric pressure ionization mass spectrometry quantitation step.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1981

Sampling methods for pharmacokinetic studies in the mouse.

Pharmacokinetics data on 2,4,5-T are used to illustrate design considerations, agent administration and sampling techniques required for single-animals studies in mice. Detailed methods for intravenous and intragastric delivery of chemicals are described, as are small volume (3 microliters) blood sampling techniques from tail vein and orbital plexus. A mouse metabolism cage is described that prohibits feed contamination of urine and feces, and also effectively segregates these elimination products. Techniques for blood, urine, and feces sample preparation and quality control assessments are also presented. Multi-sample blood concentration-time curves obtained from a single mouse are compared to data obtained using a single-sample design using may mice. While a single-animal, multiple-sample design requires the availability of microanalytical methods, it has many economic and practical advantages and results in a more accurate pharmacokinetic profile of the chemical in mice.

2,4,5-Trichlorophenoxyacetic Acid↗
PubMed1981

Small animal traversing gamma counter.

A whole body traverser was developed that provides a variety of radionuclide detection configurations for retention and distribution studies. Data from biologically incorporated radionuclides were shown to be less susceptible to geometrical problems with this whole body traverser than with the usual forms of whole body counters. When used in its fully rotational mode, the effects of photon energy and attenuation due to various body tissues, thicknesses, and radionuclide locations were minimized. Consistent presentation of all aspects of the sample produced greater than 95% correlation with the predictable data and less than 2% counting fluctuations. The whole body traverser was utilized in radiolabeled microsphere tracer studies for the determination of regional blood flow and as a static counter for long term retention studies.

Aging↗
PubMed1981

An avian tumor virus promoter directs expression of plasmid genes in Escherichia coli.

A sequence in the long terminal repeat (LTR) of avian tumor virus (ATV) DNA was shown to contain a promoter acute in Escherichia coli. For this analysis the bacterial promoters for the tetracycline (Tc) and neomycin (Nm) resistance genes were deleted from different plasmids and replaced with various fragments derived from the ATV DNA. Expression of the drug-resistant phenotype in the recombinant plasmids at levels comparable to or greater than those found with parental bacterial promotes was shown to be dependent on the presence of an intact sequence ranging from nucleotide +19 to -23 (relative to the cap site) in the ATV DNA. Comparison of the consensus bacterial promoter with the nucleotide sequence in this region revealed strong similarities.

Avian Sarcoma Viruses↗
PubMed1980

Complete nucleotide sequence of the influenza A/PR/8/34 virus NS gene and comparison with the NS genes of the A/Udorn/72 and A/FPV/Rostock/34 strains.

The nucleotide sequence of the NS gene of the human influenza virus A/PR/8/34 was determined and found to be the same length (890 nucleotides) as the NS gene of another human influenza virus A/Udorn/72 and of the avian isolate A/FPV/Rostock/34. Comparison of the sequences of the NS genes of the two human influenza viruses shows an 8.9% difference whereas the NS gene of the avian isolate differs by only 8% from that of the human strain A/PR/8/34. The extensive sequence similarity among these three genes does not support the notion of species specific homology groups among NS genes of avian and human influenza virus strains. The primary sequence of the A/PR/8/34 NS gene is consistent with the findings that the influenza virus NS gene may code for two overlapping polypeptides. In addition, an open reading frame potentially coding for a polypeptide 167 amino acids in length was found in the negative strand RNA of the A/PR/8/34 virus NS gene.

Amino Acid Sequence↗
PubMed1980

High-pressure liquid chromatography analysis of salicylic acid, salicyluric acid, and gentisic acid in biological matrixes.

An assay is reported for salicylic acid and two of its metabolites, salicyluric acid and gentisic acid, in mouse and rat blood, urine, and feces and rat embryos. The procedure involves aqueous dilution of the biological sample, addition of methanol-1% acetic acid (in water) (40:60), centrifugation, and injection of the supernate onto a commerical high-pressure liquid chromatographic column. Detection is via a variable-wavelength UV detector set at 296 nm. Quick and easy sample preparation, good accuracy and precision, and sufficient sensitivity make analysis of large numbers of samples feasible.

Animals↗