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D Farley

Publications and source records attributed to D Farley.

13 recordsLinked to original sources
PubMed1991

Expression of rat 5 alpha-reductase in Saccharomyces cerevisiae.

Dihydrotestosterone (DHT) is the principle androgen in certain tissues such as the prostate. DHT is formed from testosterone by the NADPH-dependent enzyme 5 alpha-reductase (5AR). In this paper we report the expression of catalytically active steroid 5AR from the rat in Saccharomyces cerevisiae. A full length cDNA coding for 5AR was isolated from a rat liver cDNA library and fixed in frame to the signal sequence of yeast acid phosphatase. A constitutive short promoter fragment of the acid phosphatase gene (PHO5) and the PHO5 transcriptional terminator were added and the expression cassette ligated into the yeast 2 mu vector pDP34. S. cerevisiae transformed with the 5AR expression plasmid pDP34/PHO5AR exhibited about 100-fold more activity per gram wet weight than rat prostate.

3-Oxo-5-alpha-Steroid 4-Dehydrogenase↗
PubMed1989

The human neutrophil elastase gene. Analysis of the nucleotide sequence reveals three distinct classes of repetitive DNA.

DNA sequence analysis reveals the gene encoding human neutrophil elastase to be contained on a 6-kb EcoRI fragment. The gene contains five exons and closely resembles rat mast cell proteinase II and mouse adipsin in its exon structure and intron splice phase. Non-coding regions are very rich in repetitive DNA, containing seven Alu-like segments, three distinct clustered direct repeats with monomer lengths of 53 (six repeats), 23 (three repeats) and 41 (ten repeats) nucleotides, and a 200-nucleotide AT-rich region. Protein sequence analysis, inferred from the coding regions of the gene, indicates that neutrophil elastase may contain an unusual activation peptide similar to that found in the other major neutrophil serine proteinase, cathepsin G.

DNA↗
PubMed1988

Neutrophil elastase and cathepsin G: structure, function, and biological control.

When neutrophils invade inflamed areas of the body to remove either dead or foreign components they inadvertently release potent enzymes which can, if not properly controlled, cause severe damage to healthy tissue. This can lead to a myriad of diseases including emphysema, rheumatoid arthritis, and glomuerlopnephritis, all of which are really problems of abnormal connective tissue turnover due to uncontrolled protelysis by neutrophil elastase and cathepsin G. An important step in elucidating the functions of both elastase and cathepsin G has been made by virtue of the fact that the amino acid sequence of each has been determined. Furthermore, the crystal structure of one, neutrophil elastase, is now understood. With this knowledge in mind and with the potential for a similar understanding of the mechanism of action of cathepsin G, it should soon be possible to produce synthetic inhibitors of each enzyme which can act as adjunct inhibitors to those naturally circulating in the blood or present in other tissues. As a result there is great hope for reducing the severity of injury produced by these enzymes and, therefore, in decreasing the risk for development of the debilitating diseases associated with abnormal proteolysis by neutrophil proteinases.

Amino Acid Sequence↗
PubMed1988

Molecular cloning of human neutrophil elastase.

The human U937 cell line, a promonocyte-like leukemic line, has been shown to synthesize neutrophil elastase (Senior, R. M. et al. (1982) J. Clin. Invest. 69, 384-393). We have constructed a lambda gt11 cDNA library using RNA from this cell line and isolated a clone encoding part of the protein sequence of neutrophil elastase. Nucleotide sequencing indicates the clone encodes residues 108-243 (standard chymotrypsinogen numbering) of the enzyme, plus a hitherto unsuspected 20 residue-C-terminal extension of unusual structure. Comparison of the nucleotide and amino-acid sequence of elastase with the recently reported sequence of medullasin, a proteinase isolated from bone marrow, suggests they are identical.

Amino Acid Sequence↗
PubMed1987

Molecular cloning of human cathepsin G: structural similarity to mast cell and cytotoxic T lymphocyte proteinases.

Human cathepsin G is a serine proteinase with chymotrypsin-like specificity found in both polymorphonuclear leukocytes (neutrophils) and the U937 leukemic cell line. Utilizing RNA from the latter, we have constructed a cDNA library in lambda gt11 and isolated a clone which apparently codes for the complete amino acid sequence of this enzyme. Analysis of the sequence reveals homology with rat mast cell proteinase II (47%) but a greater degree of identity (56%) with a product of activated mouse cytotoxic T lymphocytes. The close relationship between the three proteins indicates similarities in substrate specificity and in biosynthesis which we predict involves removal of a two amino acid activation peptide during or just before packaging into their respective storage granules.

Amino Acid Sequence↗
PubMed1986

Chronic measurement, using a Doppler probe, of uterine artery flow in the gravid guinea-pig.

A chronic animal model is described which permits for the first time the continuous measurement of uterine artery blood flow velocity in the pregnant guinea-pig by using a miniaturized Doppler flow probe. Preliminary validation revealed that alterations in actual blood flow are directly and proportionally related to the change in the Doppler shift (r = 0.984) from 0 to 100 ml/h. The velocity signal baseline was as stable as that of systemic blood pressure. Depending upon the individual animal's flow velocity, a deviation of 2-5% from baseline was statistically significant. With experience, greater than 90% of preparations were successful and a 30-day interval was often available for study. Uterine artery flow velocity increased steadily between 45 and 55 days of gestation. Instrumentation did not result in fetal growth retardation. A reduction in flow velocity occurred during general anaesthesia using ketamine and the antianxietal xylazine. In agreement with the reports of other investigators using a different model, both hydralazine and angiotensin II increased uterine blood velocity and adrenaline reduced it.

Angiotensin II↗