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J W Irvine

Publications and source records attributed to J W Irvine.

12 recordsLinked to original sources
PubMed1997

Use of inhibitors to identify essential cysteine proteinases of Trichomonas vaginalis.

Designing cysteine proteinase inhibitors as antitrichomonal drugs requires knowledge of which cysteine proteinases are essential to the parasite. In an attempt to obtain such information, the effects of a number of cysteine proteinase inhibitors on trichomonad growth in vitro and proteinase activity were investigated. The broad specificity inhibitor trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (known as E-64) had little effect on growth of Trichomonas vaginalis (27% inhibition at 280 microM, none at 28 microM) even though the addition of 2.8 microM E-64 to growth medium resulted in inhibition of all but two (apparent molecular masses: 35 k and 49 k) of the parasite's proteinases detected by gelatin SDS-PAGE. This shows that many of the parasite's cysteine proteinases are not essential for growth in axenic culture. In contrast, a peptidyl acyloxymethyl ketone, N-benzoyloxycarbonyl-Phe-Ala-CH2OCO-(2,6,-(CF3)2)Ph, at 16 microM killed T. vaginalis and severely inhibited growth of Tritrichomonas foetus. Exposure of Trichomonas vaginalis to 16 microM of this compound for 1 h resulted in both the 35 kDa and 49 kDa proteinases being inhibited, whereas some other proteinases were unaffected. Similar distinctions between the inhibitor sensitivity of the parasite's cysteine proteinases were apparent when a biotinylated peptidyl diazomethyl ketone was used to detect active proteinases. These data suggest that the growth inhibitory effects of the peptidyl acyloxymethyl ketone are through inhibition of cysteine proteinases that are not affected when the parasites are grown in the presence of E-64. At least one of these enzymes, which include the 35 kDa and 49 kDa cysteine proteinases, must be essential and so a suitable target for chemotherapeutic attack.

Animals↗
PubMed1993

Purification of cysteine proteinases from trichomonads using bacitracin-Sepharose.

Bacitracin affinity chromatography has been used to purify proteinases of the parasitic protozoon Tritrichomonas foetus. It proved superior to other affinity chromatography methods we have tested for the purification of trichomonad proteinases and should prove a useful procedure for purifying cysteine proteinases from these parasites and other parasitic protozoa. The main cysteine proteinases of T. foetus were purified over 100-fold to be free from the majority of other cell proteins. About 90 micrograms of protein containing 1.56-fold more proteinase activity than was detectable in the original cell lysate was obtained from 10(9) cells (7.2 mg protein). SDS-PAGE revealed that the eluate contained two main Coomassie blue-staining bands. N-terminal amino acid sequence analysis of these proteins confirmed that one of them was a cysteine proteinase with unusual features. Cysteine proteinases were also purified from cell lysates of Trichomonas vaginalis and a N-terminal sequence determined. This is the first amino acid sequence information that has been obtained for trichomonad cysteine proteinases. The method was also used to purify proteinases from the medium of T. foetus cultures. Some selectivity in binding of the proteinases to the affinity column was found.

Amino Acid Sequence↗
PubMed1993

Excimer photorefractive keratectomy for myopia.

PURPOSE: To study the safety, effectiveness, predictability, and stability of excimer laser photorefractive keratectomy (PRK) in 133 normally sighted eyes. METHODS: An excimer laser was used with a fluence of 160 mJ/cm2 at a frequency of 5 Hz and an ablation zone of 5.0 mm. The effects of nitrogen purge at surgery and postoperative steroids were evaluated. RESULTS: No significant complications occurred in any patient. After an initial over-correction, the refraction stabilized. The average results obtained at 6 months were maintained on successive examinations to 36 months. Analysis of their visual acuity in groups IIA, IIB, and III indicates that results at 6 months are predictive of final results at 1 to 3 years (P < 0.0001; r > 0.9). In myopia (1.00-6.00 diopters [D]) treated with the excimer laser, there was a trend toward improvement in results over the course of the study. In 60% in group IIA, 58% in group IIB, 71% in group III, and 88% in group III no nitrogen (no N2), +/- 1 D was obtained. For 20/40 or better uncorrected visual acuity, the outcome was 70% for group IIA, 67% for group IIB, 75% for group III, and 100% for group III no N2. Significant improvement was noted without nitrogen purge. No significant improvement was observed from steroid treatment as used. CONCLUSION: In reducing myopia, PRK appears to be safe and effective. The results obtained are reasonably predictable and stable after 6 months. As more refinements are introduced, this procedure could become one of the most promising in refractive surgery.

Adult↗
PubMed1992

Cystatin-like cysteine proteinase inhibitors of parasitic protozoa.

A new method has been developed for detecting cystatins and other cysteine proteinase inhibitors. The method, which involves protein separation by SDS-PAGE followed by a cysteine proteinase overlay step, is more sensitive than previously reported techniques: as little as 1 ng of recombinant human cystatin C can be detected and cysteine proteinase inhibitors could also be detected in complex protein mixtures such as bovine foetal serum. The method has been used to show, for the first time, cysteine proteinase inhibitors in lysates of a range of parasitic protozoa (Trypanosoma brucei, Leishmania mexicana mexicana, Toxoplasma gondii and Tritrichomonas foetus) and to confirm that one occurs in the free-living ciliate Tetrahymena pyriformis. Cystatin-like inhibitory activity was also demonstrated in boiled lysates of L. mexicana mexicana using conventional assays methods.

Animals↗
PubMed1992

Characterization of proenkephalin-cleaving proteinases in bovine adrenal chromaffin granules using [35S]proenkephalin copolymerized into sodium dodecyl sulfate-polyacrylamide gel electrophoresis.

Proteinases capable of cleaving proenkephalin into smaller peptides have been identified in bovine adrenal chromaffin granules using [35S]methionine-labeled recombinant rat proenkephalin as a selective substrate in sodium dodecyl sulfate-polyacrylamide gel electrophoresis proteinase radiozymography. This technique was used for the screening of subcellular fractions, general characterization of pH optima, and the mechanistic characterization of proteinases with both reversible and irreversible inhibitors. Two enzymes with approximate molecular masses of 76 and 30 kDa were shown to be localized to the highest-density fractions of chromaffin granules by sucrose density gradient fractionation. Both were enriched in a 1 M NaCl wash of purified chromaffin granule membranes, were active at high pH, and were characterized as serine proteinases based on inhibition by soybean trypsin inhibitor. The 30-kDa enzyme was also inhibited by diisopropyl fluorophosphate, D-Phe-Pro-Arg-CH2Cl, and D-Val-Phe-Lys-CH2Cl and appeared to be the previously described adrenal trypsin-like enzyme. A third enzyme, of 66 kDa, was also associated with the 1 M NaCl wash of purified chromaffin granule membranes but was not localized exclusively to chromaffin granules in sucrose gradients. This proteinase was found to be Ca2+ activated and inhibited by EDTA but not diisopropyl fluorophosphate, soybean trypsin inhibitor, p-chloromercuriphenylsulfonic acid, 1,10-phenanthroline, or pepstatin.

Adrenal Glands↗
PubMed1991

Partial purification and characterization of a putative prohormone-processing enzyme complex from bovine pituitary.

A putative prohormone-processing enzyme complex with specificity toward basic residues was partially purified from whole bovine pituitary glands. The complex is basic, binding to S-Sepharose at pH 8.2. The pH optimum of the enzyme is around 8.0. The enzyme is capable of cleaving proenkephalin and is present in at least three forms with relative molecular masses of about 36,000, 58,000, and 90,000 Da. The proteinase complex is inhibited by soybean trypsin inhibitor, limabean trypsin inhibitor, and aprotinin, but not by inhibitors of thiol proteinases or metal chelators. Our results indicate that this proteinase is a trypsin-like serine esterase with properties appropriate to that of a prohormone-processing enzyme.

Animals↗
PubMed1990

Electrophoretic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates: application to proenkephalin processing enzymes.

A novel method is described for the zymographic analysis of proteinases in sodium dodecyl sulfate-polyacrylamide gels containing copolymerized radiolabeled protein substrates such as [35S]methionine-labeled proenkephalin or 125I-labeled proinsulin. After electrophoresis the enzyme is reactivated and cleaves the radiolabeled in situ substrate into smaller peptides. These small peptides are able to diffuse out of the gel, leaving clear areas against a dark background when visualized by autoradiography. The technique can be used to detect as little as 200 fg of trypsin using only 50 ng (1.25 microCi) of [35S]proenkephalin. Soluble- and membrane-bound adrenal trypsin-like enzyme were isolated from bovine adrenal chromaffin granules. Both proteinases cleaved [35S]methionine-labeled proenkephalin but not 125I-labeled proinsulin. Moreover, both had a Mr of approximately 30,000. The potential of this technique for general use is discussed. An additional method using the synthetic fluorogenic substrate t-butoxycarbonyl Glu-Lys-Lys aminomethylcoumarin is also described.

Adrenal Medulla↗
PubMed1990

Turnover of sarcoplasmic proteins in the breast muscle of rapidly growing chicks.

1. Turnover of the sarcoplasmic proteins aldolase, phosphoglycerate mutase, lactate dehydrogenase and creatine phosphokinase isolated from chicken breast muscle was investigated using a pulse labelling technique. 2. A single injection of [U-14C]leucine was given and the proteins were extracted and purified at 2, 6, 15, 30, 48 and 72 hr following administration. Specific radioactivity in all of these isolated enzymes showed unexpected multiple peak profiles which did not intersect with the specific radioactivity profile of the blood plasma. 3. These results were interpreted as showing that either a large proportion of these proteins was not turned over in rapidly growing muscle or that the plasma amino acid pool was not the precursor pool for muscle protein synthesis. 4. The results also suggested that at least two sub-populations of the proteins exist within the muscle tissue. 5. A further conclusion drawn from these data was that established techniques of pulse labelling may seriously overestimate the rate of protein synthesis in growing muscle.

Animals↗