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Biomedical subjects

K A Johnson

Publications and source records attributed to K A Johnson.

At least 19 recordsLinked to original sources
PubMed1992

Mechanism and fidelity of HIV reverse transcriptase.

We have examined the RNA-dependent and DNA-dependent polymerase and ribonuclease H catalytic activities of human immunodeficiency virus reverse transcriptase using rapid transient kinetic methods with defined synthetic 25/45-mer DNA/RNA and DNA/DNA primer/templates. The Kd value for interaction of the enzyme with duplex DNA was 4.7 nM, and the value for RNA/DNA heteroduplex was of similar magnitude. A pre-steady state burst of nucleoside triphosphate incorporation was observed for both DNA and RNA templates. Analysis of the dATP concentration dependence of the burst rate provided Kd values for dATP of 4 and 14 microM and maximum rates of single nucleotide incorporation, kpol, of 33 and 74 s-1, for DNA and RNA templates, respectively. Subsequent turnovers were limited by the rate of dissociation of the primer/template from the enzyme at rates of 0.18 and 0.06 s-1 for duplex DNA and RNA/DNA heteroduplex, respectively. Analysis of rates of DNA polymerization and RNA cleavage using the RNA template revealed that the two activities are independent of one another. The polymerization rate (4-70 s-1) was dependent on dATP concentration, whereas the RNA cleavage occurred at a constant rate of 10 s-1 over the 100-fold dATP concentration range (2-200 microM). Examination of the RNA cleavage products resulting from a single turnover indicates that the polymerase and ribonuclease domains of the enzyme are separated by a distance corresponding to 19 bases of RNA/DNA heteroduplex, consistent with the recently published crystal structure (Kohlstaedt, L. A., Wang, J., Friedman, J., Rice, P. A., and Steitz, T. A. (1992) Science 256, 1783-1790). Analysis of the kinetics of processive synthesis suggested that the initial binding of dNTP leads to a faster rate of dissociation of DNA from the enzyme. Further investigation supported a two-step dNTP binding mechanism with the formation of an initial E.DNA.dNTP complex followed by a more stable E'.DNA.dNTP complex. The Kd values for incorporation of incorrect nucleoside triphosphates opposite a DNA template thymidine were 1010 microM for dGTP, 1240 microM for dCTP, and 840 microM for dTTP. The corresponding maximum kpol rates were 4.8 s-1 for dGTP, 0.52 s-1 for dCTP, and 0.41 s-1 for dTTP. These values provide fidelity estimates of 1740 for discrimination against dGTP, 19,700 for dCTP, and 16,900 for dTTP misincorporations at this site.

Base Sequence↗
PubMed1992

Dynamics of ribozyme binding of substrate revealed by fluorescence-detected stopped-flow methods.

Fluorescence-detected stopped-flow and equilibrium methods have been used to study the mechanism for binding of pyrene (pyr)-labeled RNA oligomer substrates to the ribozyme (catalytic RNA) from Tetrahymena thermophila. The fluorescence of these substrates increases up to 25-fold on binding to the ribozyme. Stopped-flow experiments provide evidence that pyr experiences at least three different microenvironments during the binding process. A minimal mechanism is presented in which substrate initially base pairs to ribozyme and subsequently forms tertiary contacts in an RNA folding step. All four microscopic rate constants are measured for ribozyme binding of pyrCCUCU.

Animals↗
PubMed1992

Cloning and expression of gene 4 of bacteriophage T7 and creation and analysis of T7 mutants lacking the 4A primase/helicase or the 4B helicase.

T7 gene 4, which is required for DNA replication, specifies two proteins whose coding sequences overlap in the same reading frame: the 4A protein, a 566-amino acid primase/helicase, and the 4B protein, a 503-amino acid helicase whose initiation codon is the 64th codon of the 4A protein. To study better the individual functions of these two overlapping proteins, we made clones that express both 4A and 4B proteins, only 4B protein, or only what we refer to as the 4A' protein, in which methionine 64 is replaced by leucine, thereby eliminating the 4B initiation codon. These clones provide considerably more gene 4 protein for biochemical analysis than do infected cells. They can also be used to isolate and propagate T7 gene 4 deletion mutants, and we have made T7 mutants which lack all gene 4 coding sequences, or which express 4A' protein but no 4B protein, or 4B protein but no 4A protein. Analysis of these phage mutants shows that 4A' protein without any 4B protein can support essentially normal replication and growth, whereas 4B protein without any 4A protein supports little replication or growth. Apparently, the primase activity of the 4A protein is essential for replication, but the 4B protein is dispensable, presumably because the 4A protein also supplies helicase activity. The mutation at amino acid 64 of 4A' appears to have little effect on 4A function. The rate of replication during normal T7 infection appears to be limited by the amount of gene 4 protein, but too high a level of either 4A or 4B protein is inhibitory to growth.

Base Sequence↗
PubMed1992

Large scale purification and biochemical characterization of T7 primase/helicase proteins. Evidence for homodimer and heterodimer formation.

A rapid purification procedure produces milligram amounts of the T7 gene 4A' primase/helicase, 4B helicase, and the wild-type 4AB proteins expressed from the clones described in the accompanying paper (Rosenberg, A. H., Patel, S. S., Johnson, K. A., and Studier, F. W. (1992) J. Biol. Chem. 267, 15005-15012). Purified 4A' protein (in which the wild-type methionine at amino acid 64 has been replaced by leucine to eliminate the 4B initiation codon) appears to be equivalent to the wild-type 4A protein in primase, helicase, and NTPase activities. Gel filtration chromatography and polyacrylamide gel electrophoresis of native proteins indicate that the 4A' and 4B proteins form homodimers and heterodimers in solution. Heterodimer formation presumably accounts for an observed 3-fold increase in the primase activity of 4A' upon addition of 4B that lacks primase activity of its own. Steady-state k(cat) and Km values for hydrolysis of the nucleoside triphosphates ATP, dATP, dTTP, and dGTP were measured for 4A', 4B, 4A'B (1:1), and wild-type 4AB (1:2) proteins. The dependence of the dNTPase activities on the concentration was hyperbolic, suggesting single or noncooperative binding sites, whereas ATPase activity was sigmoidal, suggesting more than one ATP binding site. The k(cat)/Km ratios for hydrolysis of the dNTPs by the four protein preparations were within a factor of 6 of each other. The 1:1 mixture of 4A'B had the highest k(cat)/Km ratios, with a preference for dATP and dTTP.

Cell Line↗
PubMed1992

Conformational coupling in DNA polymerase information transfer.

The extraordinary fidelity of DNA replication during forward polymerization and exonuclease error correction is largely a function of a conformational change that occurs in response to a correct dNTP binding to properly base-paired duplex DNA. The conformational change serves as a kinetic barrier to effect the rapid incorporation of correct bases while minimizing the rate of polymerization with incorrect bases and allowing for selective removal of mismatches. However, in spite of the number of attractive features to the conformational change model, the evidence in support of such a rate-limiting step is still subject to significant uncertainty. It is the challenge of further work on DNA polymerases as well as many other enzyme systems to devise new methods to define the transient state of the enzyme during catalysis and to relate the kinetic and thermodynamic parameters to the enzyme structure.

Base Sequence↗
PubMed1992

Effect of protein tyrosine phosphatase 1B expression on transformation by the human neu oncogene.

Many oncogenes encode proteins with a tyrosine kinase activity that appears to be directly involved in the process of transformation. Because these kinases are themselves activated for transformation by tyrosine phosphorylation, proteins which remove phosphate from tyrosine residues, protein tyrosine phosphatases (also termed phosphotyrosine phosphatases and protein phosphotyrosyl phosphatases), are intuitive candidate transformation suppressors. The human PTP1B gene, previously cloned in our laboratory and encoding the low molecular weight protein tyrosine phosphatase PTPase 1B, was introduced into NIH 3T3 cells. Subsequent transformation of these PTPase 1B-expressing cells by an oncogenic form of the human neu gene was suppressed relative to control NIH 3T3 cells. This suppression of transformation was observed in assays for focus formation, anchorage-independent growth, and tumorigenicity. Tumorigenicity assays indicated a complex effect of PTPase 1B expression on transformation.

3T3 Cells↗
PubMed1992

Replication of basal bodies and centrioles.

During the past year, studies on the centrioles and basal bodies of animal and algal cells, and the spindle pole bodies of yeast and other fungi, have added significantly to our knowledge of how these cell organelles form and how they function in initiating microtubule assembly throughout the cell cycle. Most of these studies have used antibodies to identify proteins within and around these organelles and, in some cases, to disrupt their ability to nucleate microtubules. Genetic methods have been used to identify specific proteins, including a new member of the tubulin superfamily, involved in the function and replication of spindle pole bodies and centrioles.

Animals↗
PubMed1992

Musculoskeletal and soft tissue Aeromonas infection: an environmental disease.

During a 4-year period from November 1985 to November 1989, Aeromonas was isolated from wounds and soft tissues with clinical evidence of infection in 28 patients at our institution. Of the 28 patients, 23 (82%) had sustained an acute open or penetrating injury, more than half of which (13 of the 23) were water-related trauma. One patient had Aeromonas osteomyelitis. Five patients had no history of trauma, and three of these five had an underlying chronic disease. Treatment included débridement and antimicrobial agents. Susceptibility testing on 25 isolates from 23 patients showed uniform resistance to ampicillin and considerable resistance to cefazolin sodium (68%), but all isolates were sensitive to gentamicin sulfate, cefuroxime sodium, and the third-generation cephalosporins.

Academic Medical Centers↗
PubMed1992

Polarity of flagellar assembly in Chlamydomonas.

During mating of the alga Chlamydomonas, two biflagellate cells fuse to form a single quadriflagellate cell that contains two nuclei and a common cytoplasm. We have used this cell fusion during mating to transfer unassembled flagellar components from the cytoplasm of one Chlamydomonas cell into that of another in order to study in vivo the polarity of flagellar assembly. In the first series of experiments, sites of tubulin addition onto elongating flagellar axonemes were determined. Donor cells that had two full-length flagella and were expressing an epitope-tagged alpha-tubulin construct were mated (fused) with recipient cells that had two half-length flagella. Outgrowth of the shorter pair of flagella followed, using a common pool of precursors that now included epitope-tagged tubulin, resulting in quadriflagellates with four full-length flagella. Immunofluorescence and immunoelectron microscopy using an antiepitope antibody showed that both the outer doublet and central pair microtubules of the recipient cells' flagellar axonemes elongate solely by addition of new subunits at their distal ends. In a separate series of experiments, the polarity of assembly of a class of axonemal microtubule-associated structures, the radial spokes, was determined. Wild-type donor cells that had two full-length, motile flagella were mated with paralyzed recipient cells that had two full-length, radial spokeless flagella. Within 90 min after cell fusion, the previously paralyzed flagella became motile. Immunofluorescence microscopy using specific antiradial spoke protein antisera showed that radial spoke proteins appeared first at the tips of spokeless axonemes and gradually assembled toward the bases. Together, these results suggest that both tubulin and radial spoke proteins are transported to the tip of the flagellum before their assembly into flagellar structure.

Animals↗
PubMed1992

The effects of consultation on over-the-counter medication purchasing decisions.

This article examines factors that predict changes in consumer purchasing decisions of nonprescription medications. Variables corresponding to factors in Andersen's behavioral model are measured, in addition to data regarding characteristics of the 17 pharmacy consultants who provided counseling services. One thousand seven hundred and thirteen consumers in five stores in southern California were provided consultation during a 6-month period, resulting in 25.4% of the patients purchasing a different drug than intended when entering the pharmacy, 1.3% being referred to a physician, and 13.4% not purchasing any over-the-counter medication at all. Logistic regression techniques demonstrated that one enabling variable (availability of generic medications), and four need factors (the discussion of clinical issues, short encounters, cough and cold products, and vitamin products) were significant predictors of the consumer's decision to purchase a different product than intended. Consultant characteristics (introversion, external locus of control) were also important predictors, but opposite the expected direction. Consumers who received information from female consultants were more likely to change their purchasing decisions.

Adult↗
PubMed1992

Fetal alcohol syndrome: a case report of neuropsychological, MRI and EEG assessment of two children.

Neuropsychological, neuroanatomical, and electrophysiological data are presented on two subjects with fetal alcohol syndrome (FAS). Both boys had intelligence quotients in the mentally deficient range and were found to have several other severe, specific deficits. Magnetic resonance imaging showed abnormalities of the corpus callosum, and reductions in the size of the basal ganglia and thalamic structures. No focal abnormalities were noted in the electroencephalogram records, although the electroencephalograms of both boys were moderately abnormal for their age group. A multidisciplinary approach to the study of FAS, hopefully will lead to a more unified concept of the disorder and perhaps indicate specific areas of vulnerability.

Adolescent↗
PubMed1992

Acute toxicity in rats of chlorinated hydrocarbons given via the intratracheal route.

1. The approximate lethal dose (ALD) of six chlorinated hydrocarbons via the intratracheal route has been determined in rats and compared with published oral LD50 values. 2. The compounds tested in this study were dichloromethane, perchloroethylene, trichloroethylene, carbon tetrachloride, chloroform and ethylene dichloride. 3. A method of administering the materials intratracheally to unanaesthetized animals was developed. 4. The intratracheal ALD of the chlorinated hydrocarbons ranged from 3.1 to 17.5% of the oral LD50 and death was peracute. 5. Aspiration of chlorinated hydrocarbons may present more of a hazard than oral toxicity and should be considered when rendering first aid or emergency medical treatment.

Administration, Oral↗
PubMed1992

The patellar tendon-bearing brace as treatment for neurotrophic arthropathy: a dynamic force monitoring study.

UNLABELLED: The effects of shoe wear, custom-made inserts, patellar tendon-bearing (PTB) braces, and extra-padded PTB braces on load transmission to neuroarthropathic (Charcot) feet of six diabetic patients were assessed. A PTB brace with a free ankle and an extradepth shoe without external modifications was used. Four distinct anatomical regions were evaluated by an in-shoe technique measuring vertical force. The mean peak force transmitted to the entire foot was higher in the affected than in the unaffected extremity barefoot and shod. The mean peak forces under the midfoot were higher for the affected than for the unaffected extremity for all tested circumstances. In the affected foot, use of the standard PTB brace reduced mean peak force to the entire foot by 15%. Adding extra padding to the brace decreased the mean peak force 32%. The effect of padding on vertical load transmission was greatest for patients who had worn the brace the longest. For the patients who had worn it longer than average, the addition of padding decreased the mean peak force to the entire foot by 19%, to the hindfoot by 37%, and to the midfoot by 20%. CLINICAL RELEVANCE: In this preliminary study, we found that a properly fitted PTB brace can reduce load transmission to the Charcot foot. Specifically, load transmission was reduced to the hindfoot, but not to the midfoot or forefoot. Based on these results, we cannot recommend its use to reduce vertical force transmission to the Charcot midfoot or forefoot. Long-term PTB brace use, especially in the limited weight-bearing patient, should be regularly adjusted to ensure adequate brace fit.

Aged↗
PubMed1992

Molecular analysis of the Neurospora clock: cloning and characterization of the frequency and period-4 genes.

Genetic analysis of Neurospora crassa has identified many mutants that affect the biological clock. In this article we review the cloning of two of these genes, frq and prd-4. Both genes were isolated using a chromosome walk technique. Subcloning experiments and subsequent Northern analysis of frq implicate the importance of two transcripts that emanate from this locus. In preliminary data, no protein-coding region is evident in the smaller transcript; the larger transcript contains a 962-amino acid open reading frame. The open reading frame shows limited homology to per, a clock gene identified in Drosophila. Sequence analysis of all existing frq alleles suggests that the defect in each case lies within the open reading frame. Successful cloning of the prd-4 gene required walking a distance of greater than 40 kb. A physical map of this region has been constructed using restriction analysis. The dominance-recessive relationship of prd-4 and prd-4+ was established by examining the period lengths of strains harboring a wide range of prd-4/prd-4+ nuclear ratios.

Amino Acid Sequence↗
PubMed1992

The drinking-smoking syndrome and social context.

Drinking and smoking concurrence is examined in a national sample of 6,072 respondents. Results indicate that the two addictive behaviors are synergistically associated in the general population: persons who both drink and smoke tend to drink to a greater extent than nonsmokers; drinkers smoke more than nondrinkers and smokers drink more than nonsmokers. As predicted, a multivariate analysis of drinking and smoking covariance was significantly correlated with social group affiliation, both across the life span and proportionately among men and women. The social context of interpersonal relationships may therefore be a critical factor in the process of pharmacological conditioning and environmental reinforcement of the drinking and smoking habits. This report confirms the strong drinking and smoking covariation found among social drinkers and heavy alcohol consumers in clinical and experimental studies, suggesting that drinking and smoking may also be interrelated in the rehabilitative process. Finally, it is suggested that the present investigation extends the research literature on the drinking and smoking syndrome in several important respects. Various implications of the study are discussed.

Adolescent↗
PubMed1992

Maintenance energy requirement of llamas.

Five castrated male llamas (mean body weight, 94 kg) were studied in an energy balance trial to determine maintenance energy requirement of llamas. Llamas were fed a 50% oat hay-50% pelleted concentrate diet (2.43 Mcal of metabolizable energy/kg of diet dry matter) at approximately 1.6% of body weight (BW). An 8-day total collection digestion trial was used to determine fecal and urine energy losses. Heat production and methane emissions were determined via indirect respiration calorimetry measurements on each llama fed at the same level of intake as during the digestion trial and subsequently on days 3 and 4 of a period of nonfeeding. Fecal, urine, and methane energy losses of the llamas fed near-maintenance intake were 32.5, 3.5, and 7.1% of gross energy intake, respectively. The postabsorptive metabolic rate, commonly called nonfed (fasting) heat production, was 59.3 kcal/BW0.75. Using a linear relation between postabsorptive and maintenance energy requirement and efficiency of energy use below maintenance of 0.702, metabolizable energy requirement at maintenance was determined to be 84.5 kcal/BW0.75.

Animal Feed↗
PubMed1992

An approach to evaluating heuristics in abduction: a case study using RedSoar--an abductive system for red blood cell antibody identification.

Abduction, or inference to a best explanation, is a ubiquitous type of inference that is frequently used by humans in a wide range of tasks. However, many realistic domains have properties that make abduction computationally intractable (i.e., where the time to reach a solution increases exponentially with the number of possible explanations). We present a domain task analysis and performance evaluation of RedSoar, a plausible cognitive computational model of abduction, that accomplishes the antibody identification task in immunohematology. The task analysis reveals how a computationally intractable abductive problem, where one is seeking optimal solutions, can be reformulated to be a computationally tractable abductive problem, by seeking satisfactory rather then optimal solutions. From the satisfactory perspective, our evaluation framework of RedSoar's performance explores the computational benefits and costs of having directly available abstract hypothesis formation knowledge, and how a strong causal constraint between hypotheses and data reduces the combinatorial explosion of constructing a best explanation.

Antibodies↗