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J M McCoy

Publications and source records attributed to J M McCoy.

At least 19 recordsLinked to original sources
PubMed1995

Alanine-scanning mutagenesis of human interleukin-11: identification of regions important for biological activity.

We have identified functionally important regions of human interleukin-11 (hIL-11) by means of alanine-scanning mutagenesis. A total of 61 mutated forms of hIL-11 were produced in E. coli as thioredoxin fusion proteins and tested in a murine T10 plasmacytoma proliferation assay. Mutations made at several positions proximal to the hIL-11 C-terminus caused substantial reduction in biological activity. In addition a number of other mutations in this region affected either protein folding or stability. Both effects displayed a characteristic periodicity with respect to the primary sequence which suggested that residues close to the C-terminus of hIL-11 adopt a helical conformation. Mutations made proximal to the N-terminus of hIL-11 also exhibited reduced bioactivity, although no effects on protein folding or stability were observed. The N-terminal mutations with reduced activity also mapped with a periodicity suggestive of a helical conformation. We previously have proposed a four-helix bundle topology for the hIL-11 structure based on physical studies, selective chemical modifications, positions of intron/exon boundaries, limit proteolysis experiments and site-directed mutagenesis. The alanine-scanning mutagenesis data we report here provide additional support for this model.

Alanine↗
PubMed1995

Structure-function relationships in human interleukin-11. Identification of regions involved in activity by chemical modification and site-directed mutagenesis.

Chemical modification approaches combined with site-directed and deletion mutagenesis have been used to identify functionally critical regions/residues of recombinant human IL-11 (rhIL-11). Incubation of rhIL-11 with iodoacetic acid results in specific alkylation of a single methionine residue, Met58, and a 25-fold reduction of in vitro biological activity on mouse plasmacytoma cells. A similar decrease in activity is observed when Met58 is substituted with Ala, Leu, Gln, Glu, or Lys by site-directed mutagenesis. Treatment of rhIL-11 with succinic anhydride leads to modification of the amino-terminal amino group and partial labeling of 2 lysines, Lys41 and Lys98, and to a 3-fold decrease in activity. The activity losses can be attributed to modification of the lysine residues, since the succinyl derivative of the amino terminus is fully active. In addition, carboxyl-terminal deletion mutagenesis studies have demonstrated that removal of the last 4 residues reduces rhIL-11 activity 25-fold, whereas removal of 8 or more amino acids results in an inactive molecule. Based on secondary structure predictions and the location of exon/intron boundaries in the IL-11 genomic structure, we propose a four-helix bundle topology as a structural model for rhIL-11. This model has been tested by limited proteolysis using three side chain-specific endoproteinases. A limited number of protease-sensitive cleavage sites are present in rhIL-11, and all but two are located in the postulated helix interconnecting loops or at helix termini. alpha-Helices, which in the proposed structure form a compact core of the molecule, are inaccessible to digestion under limiting conditions. According to the model, Met58, Lys41 and Lys98 are located on the surface of the molecule, in agreement with their preferential accessibility to chemical modifications. By analogy with human growth hormone, we postulate that Met58 and the carboxyl terminus of rhIL-11 are involved in the primary receptor binding site (site I), whereas Lys41 and Lys98 may be a part of binding site II.

Amino Acid Sequence↗
PubMed1995

Gene fusion expression systems in Escherichia coli.

In recent years, Escherichia coli gene fusion expression systems have circumvented many of the problems inherent in the use of this bacterium for the production of recombinant proteins. These systems also provide a powerful means for identifying peptides or proteins with desired binding specificities. Gene fusion technology continues to expand with the introduction of new fusion partners, purification and detection tags, cleavage reagents and ways to display peptides on the surface of bacteria.

Cloning, Molecular↗
PubMed1993

A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm.

We have developed a versatile Escherichia coli expression system based on the use of E. coli thioredoxin (trxA) as a gene fusion partner. The broad utility of the system is illustrated by the production of a variety of mammalian cytokines and growth factors as thioredoxin fusion proteins. Although many of these cytokines previously have been produced in E. coli as insoluble aggregates or "inclusion bodies", we show here that as thioredoxin fusions they can be made in soluble forms that are biologically active. In general we find that linkage to thioredoxin dramatically increases the solubility of heterologous proteins synthesized in the E. coli cytoplasm, and that thioredoxin fusion proteins usually accumulate to high levels. Two additional properties of E. coli thioredoxin, its ability to be specifically released from the E. coli cytoplasm by osmotic shock or freeze/thaw treatments and its intrinsic thermal stability, are retained by some fusions and provide convenient purification steps. We also find that the active-site loop of E. coli thioredoxin can be used as a general site for small peptide insertions, allowing for the high level production of soluble peptides in the E. coli cytoplasm.

Amino Acid Sequence↗
PubMed1991

The structure of expert diagnostic knowledge in occupational medicine.

Development of an artificial intelligence expert system for diagnosing occupational lung disease requires explicit specification of the structure of knowledge necessary in clinical occupational medicine independent of the process by which the knowledge is utilized. Furthermore, explicit recognition of sources of uncertainty is necessary. Seven categories of knowledge define the diagnostic knowledge base in occupational pulmonary medicine. These include four objects (jobs, industries, exposures, and diseases) and three relationships between pairs of objects. This analysis demonstrates some of the unique aspects of occupational medicine expertise.

Algorithms↗
PubMed1991

Status report on ambulatory care and education in the VA Western Region and western medical schools.

Since the initial 1988 Ambulatory Care and Education (ACE) conference, reported in the October 1989 supplement to Academic Medicine, the Western Region Veterans Health Administration and its 11 affiliated western medical schools have established several programs and related activities that implement strategic ACE recommendations. This report gives an update on the ACE Advisory Group; the Pilot Ambulatory Care and Education (PACE) Center at the VA Medical Center, Sepulveda, California; other innovative ambulatory care and education projects; the second ACE Development Conference; and future activities in the expanded Western Region of the Department of Veterans Affairs.

Ambulatory Care↗
PubMed1991

Artificial intelligence-assisted occupational lung disease diagnosis.

An artificial intelligence expert-based system for facilitating the clinical recognition of occupational and environmental factors in lung disease has been developed in a pilot fashion. It utilizes a knowledge representation scheme to capture relevant clinical knowledge into structures about specific objects (jobs, diseases, etc) and pairwise relations between objects. Quantifiers describe both the closeness of association and risk, as well as the degree of belief in the validity of a fact. An independent inference engine utilizes the knowledge, combining likelihoods and uncertainties to achieve estimates of likelihood factors for specific paths from work to illness. The system creates a series of "paths," linking work activities to disease outcomes. One path links a single period of work to a single possible disease outcome. In a preliminary trial, the number of "paths" from job to possible disease averaged 18 per subject in a general population and averaged 25 per subject in an asthmatic population. Artificial intelligence methods hold promise in the future to facilitate diagnosis in pulmonary and occupational medicine.

Artificial Intelligence↗
PubMed1989

Quality of care in family practice: does residency training make a difference?

As the proportion of physicians who enter residency training in family practice steadily increases, so does the need to evaluate the impact of their training and postgraduate education on the quality of care in their practices. We audited the practices of 120 randomly selected family physicians in Ontario, who were separated into four groups: nonmembers of the College of Family Physicians of Canada (CFPC), members of the CFPC with no certification in family medicine, certificated members without residency training in family medicine and certificated members with residency training in family medicine. The practices were assessed according to predetermined criteria for charting, procedures in periodic health examination, quality of medical care and use of indicator drugs. Generally the scores were significantly higher for CFPC members with residency training in family medicine than for those in the other groups, nonmembers having the lowest scores. Patient questionnaires indicated no difference in satisfaction with specific aspects of care between the four groups. Self-selection into residency training and CFPC membership may account for some of the results; nevertheless, the findings support the contention that residency training in family medicine should be mandatory for family physicians.

Adult↗
PubMed1989

Human immunodeficiency virus 1 reverse transcriptase. Template binding, processivity, strand displacement synthesis, and template switching.

We have analyzed the kinetics of DNA synthesis catalyzed by reverse transcriptase from human immunodeficiency virus 1 (HIV-1). Reverse transcriptase, overproduced in Escherichia coli and purified to homogeneity, has polymerase and RNase H activity. Reverse transcriptase forms a stable complex with poly(rA).oligo(dT) primer-templates in the absence of Mg2+ and dTTP with an equilibrium dissociation constant of 3 nM. Synthesis from these preformed complexes can be initiated, and restricted to a single processive cycle, by the simultaneous addition of Mg2+, dTTP, and excess competitor RNA. Preformed complexes decay with a maximal half-life of 2-3 min. Synthesis on poly(rA) templates is processive with an incorporation rate of 10-15 nucleotides/s at 37 degrees C. Processivity varies widely with the template used, increasing from a few to greater than 300 nucleotides in the order: poly(dA) less than double-stranded DNA less than single-stranded DNA less than single-stranded RNA less than poly(rA). On double-stranded DNA reverse transcriptase catalyzes limited strand-displacement synthesis of up to 50 nucleotides. On RNA-DNA hybrids significant DNA synthesis is observed only after degradation of the RNA strand by the RNase H activity of reverse transcriptase. Intermolecular strand switching occurs with poly(rA) templates. At low ionic strength reverse transcriptase can use multiple templates with a single primer, leading to products of greater than template length. Reverse transcriptase and primer do not have to dissociate during the exchange of template strands, thus allowing processive DNA synthesis across template borders.

DNA, Single-Stranded↗
PubMed1989

Evaluating preclinical medical students by using computer-based problem-solving examinations.

A study was designed to determine the feasibility of creating and administering computer-based problem-solving examinations for evaluating second-year medical students in immunology and to determine how students would perform on these tests relative to their performances on concurrently administered objective and essay examinations. The results indicate that problem-solving questions of uniform difficulty can be created and that correct solution of the problems correlates with the early selection of the most relevant data. The mean scores on the three types of tests were not significantly different. The scores on the essay and objective examinations were most highly correlated, those on the computer-based and objective examinations were minimally correlated, and those on the computer-based and essay examinations were not correlated.

Allergy and Immunology↗
PubMed1989

Predicate calculus, artificial intelligence, and workers' compensation.

Application of principles of predicate calculus (PC) and artificial intelligence (AI) search methods to occupational medicine can meet several goals. First, they can improve understanding of the diagnostic process and recognition of the sources of uncertainty in knowledge and in case specific information. Second, PC provides a rational means of resolving differences in conclusion based upon the same premises. Third, understanding of these principles allows separation of knowledge (facts) from the process by which they are used and therefore facilitates development of AI-based expert systems. Application of PC to recognizing causation of pulmonary fibrosis is demonstrated in this paper, providing a method that can be generalized to other problems in occupational medicine. Application of PC and understanding of AI search routines may be particularly applicable to workers' compensation where explicit statement of rational and inferential process is necessary. This approach is useful in the diagnosis of occupational lung disease and may be particularly valuable in workers' compensation considerations, wherein explicit statement of rationale is needed.

Artificial Intelligence↗
PubMed1988

Ameloblastoma.

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Adult↗
PubMed1987

Structure-function studies on bacteriorhodopsin. I. Expression of the bacterio-opsin gene in Escherichia coli.

To express the bacterio-opsin (bop) gene in Escherichia coli, we have employed the inducible expression vectors pIN-II-A, -B, and -C (Nakamura, K., and Inouye, M. (1982) EMBO J. 1, 771-775). The vectors contain three cloning sites early in the E. coli lipoprotein gene (lpp) which is transcribed from tandem lpp and lac promoters. The bop gene was modified so as to delete the N-terminal leader sequence and then cloned into each of the three cloning sites to encode three different lipoprotein/bacterio-opsin fusions. Expression of the fusions was demonstrated both in vitro and in vivo. The fusion protein was estimated to be about 0.05% of the total cell protein. The cause for the low level of expression apparently was neither an inadequate level of mRNA nor degradation of the protein. However, expression of the fusions caused inhibition of the growth of the host to varying extents. One fusion protein was purified from E. coli membranes to homogeneity by immunoaffinity chromatography followed by preparative gel electrophoresis. The purified fusion protein generated a bacteriorhodopsin-like chromophore on treatment with defined lipid/detergent mixtures and retinal. When reconstituted into vesicles, the protein pumped protons on illumination comparably to the reconstituted native bacterio-opsin.

Bacteriorhodopsins↗
PubMed1986

The nucleotide sequence of Scenedesmus obliquus chloroplast elongator methionine-accepting tRNA.

The chloroplast elongator methionine-accepting tRNA from the green alga Scenedesmus obliquus was purified and and its nucleotide sequence determined. The sequence does not bear close homology to higher-plant chloroplast tRNAMetm species, nor to the chloroplast tRNAMetm species from Euglena gracilis. This tRNA is unique among all tRNAs in having pseudouridine at position 8, and this has implications with regard to the proposed interaction of aminoacyl-tRNA synthetases with their cognate tRNAs at this position. The primary sequence of the tRNA is: pG-C-C-U-G-C-U-psi-A-G-C-U-C-A-G-U-D-Gm- G-C-C-A-G-A-G-C-A-U-C-C-G-psi-psi-U-C-A-U-m2A-C-G-C-G-G-A-A-A- m7G-D-C-A-C-U-A-G-T-psi-C-G-A-A-U-C-U-A-G-U-A-G-C-A-G-G-C-A-C-C-A-OH.

Base Sequence↗