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Biomedical subjects

J W Mitchell

Publications and source records attributed to J W Mitchell.

At least 19 recordsLinked to original sources
PubMed1998

Analysis of the D1S80 locus by capillary electrophoresis.

We have demonstrated in a systematic manner that the capillary electrophoresis (CE) method of genotyping the human D1S80 locus is an effective replacement for the commonly used gel electrophoresis method. The CE method is fast, with a total run time of less than 22 min per sample. Separation has been optimized so that resolution for all pairs of alleles from 14 through 41 is greater than 1, providing unambiguous identification. The method utilizes run buffer containing 0.30% hydroxyethyl cellulose as the sieving polymer in a 50 microm internal diameter (ID) column coated with a 0.1 microm DB-17 film. Laser-induced fluorescence (LIF) with YO-PRO-1 intercalating dye is used for detection. Internal standards of 300 and 1000 bp bracket the D1S80 region in the electropherogram. Two typing procedures were evaluated: matching of the normalized migration times of sample alleles to ladder alleles; and software alignment of sample and ladder electropherograms using the internal standard peaks as references. Using the first procedure, 91.5% of the D1S80 genotypes were unambiguous, while 100% were unambiguous using the second procedure. Seventy-nine routine samples were analyzed in a side-by-side comparison of the CE and slab gel methods, with complete agreement of the results. Twenty-two samples were selected from a large database previously analyzed by the slab gel method to demonstrate that all alleles from 14 through 41 were typed correctly, including samples containing adjacent alleles. Additionally, 43 samples containing alleles greater than the 41 repeat number were typed correctly.

Automation↗
PubMed1998

The leaching of nickel from new and used metal spectacle frames.

Fifty "used" and 373 new spectacle frames were tested for evidence of nickel release using dimethylglyoxime and ammonia. Nearly all the "used" frames (96%) tested positive. A lower proportion (24.5%) of new frames showed evidence of nickel leaching. It is suggested that this is evidence of non-conformance with the current British Standard although all our suppliers believed that their frames conformed with BS 6625 (1985).

Dermatitis, Contact↗
PubMed1997

Gold plating on spectacle frames.

An investigation was carried out into the thickness and standard of application of the plating and lacquer coatings applied to three metal spectacle frames. All conform to BS 6625 (1991) for plating thickness, but there was considerable variation in regularity and porosity.

Electroplating↗
PubMed1997

The variation in the threads of the dowel screws of spectacle frames.

British Standards specify a very small number of screw sizes as being acceptable for use in spectacle frames. We have measured the screw sizes of 50 frames, selected at random from the stock of Glasgow Caledonian University Clinic and have shown that very few of the screws used conform to these standards.

Equipment Design↗
PubMed1993

Fibrolase. A fibrinolytic protein from snake venom.

Fibrolase is an active fibrinolytic agent and possesses potential for use in thrombolytic therapy. Its mode of action had been characterized, both in vitro and in vivo. Possessing three disulfide bonds, native fibrolase is nonglycosylated and binds an intrinsic zinc atom. The zinc is essential for retention of activity and structural integrity. In solution, fibrolase is sensitive to changes in pH and temperature (Pretzer et al., 1991). At neutral to basic pH (pH 5-9), the solubility and stability of fibrolase is nearly constant. Little structural variation can be detected by CD spectroscopy. However, decrease in pH below 5 leads to a pronounced reduction in both the solubility and activity of fibrolase. At pH 3 and below, the solubility of fibrolase returns but the activity does not. This solubility profile is unusual in that the minimal solubility is well removed from the pI (which is 6.7). It is proposed that the behavior of fibrolase with variation in pH can be understood in terms of capacity to bind zinc. At pH 5 to 9, the protein binds zinc and the structure and activity are preserved. Near pH 5, the histidine residues which serve as ligands for the zinc become protonated and zinc binding is lost. Loss of zinc leads to local unfolding of a helical segment of fibrolase, exposing hydrophobic groups which allow the protein to rapidly aggregate. At lower pH values (1-3), the protein again adopts a more globular structure, similar to molten globule states, and the solubility increases. However, without the zinc, fibrolase remains inactive. Changes in pH also affect thermal stability. The Tm for fibrolase moves from 50 degrees C at pH 8 to 43 degrees C at pH 5. Increases in temperature also lead to removal of the zinc ion, again producing a partially denatured protein with a marked tendency to aggregate. In both cases (decrease in pH and increase in temperature), analysis of the CD spectra indicates that the protein has primarily lost alpha-helical secondary structure. A major change in structure can also be observed using NMR spectroscopy. At temperatures below 35 degrees C, the globular structure of fibrolase remains intact, although some increase in chain mobility can be noted with increased temperature. Upon melting, numerous signals collapse as the protein unfolds. Transition temperatures (Tm) as measured by CD and NMR are in good agreement. Similar structural changes can be induced by adding zinc chelators such as EDTA and DTT. This leads to complete loss of activity at EDTA concentrations above 1.0 mM.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1992

Effect of zinc binding on the structure and stability of fibrolase, a fibrinolytic protein from snake venom.

Fibrolase is a metalloprotease with potential use as a fibrinolytic agent. Loss of the intrinsic zinc atom leads to a rapid decrease in enzymatic activity. Circular dichroism measurements indicate that there is a partial unfolding of an alpha-helical section of the protein concomitant with the loss of zinc. Removal of zinc can be affected by elevated temperatures, acidic pH values, and addition of chelating agents. At low molar concentrations, both ethylenediaminetetraacetic acid (EDTA) and dithiothreitol (DTT) were found to remove zinc efficiently. Analysis of the sequence of fibrolase identified a segment which possessed a high degree of homology with the metal binding site of other zinc proteases, such as thermolysin and the collagenases. However, the putative zinc binding site in fibrolase lacks the additional glutamate ligand found in thermolysin and subtilisin. This sequence is also predicted to adopt an alpha-helical conformation. Together, these data indicate that there is a well-defined metal binding site in fibrolase and that metal binding is the most important factor governing the stability of this protein.

Amino Acid Sequence↗
PubMed1991

Stability of the thrombolytic protein fibrolase: effect of temperature and pH on activity and conformation.

The effect of temperature and pH on the activity and conformation of the thrombolytic protein fibrolase was examined. Fibrolase maintained proteolytic activity over 10 days at room temperature (approximately 22 degrees C). At 37 degrees C, greater than 50% of the proteolytic activity was lost within 2 days and no activity remained after 10 days. Circular dichroism (CD) spectra at elevated temperatures showed that alpha-helical structure was lost in a cooperative transition (Tm of 50 degrees C at pH 8). Structural changes were detected by NMR prior to unfolding which were not observable by CD, and the Tm determined by NMR was 46 degrees C at pD 8. The effect of pH on the proteolytic activity and structure of fibrolase was examined over the pH range from 1 to 10. Activity was maintained at neutral to alkaline pH values from pH 6.5 to pH 10.0 but decreased substantially in acidic media. While CD spectra indicated little variation in secondary structure over the pH range 5 to 9, significant differences were noted at pH 2 to 3. The melting temperature of fibrolase decreased to 43 degrees C at pH 5. Protein concentrations determined over the pH range of 1 to 10 showed an apparent solubility minimum at pH 5.0, which did not correspond to the isoelectric point of 6.5. Explanations for these observations are proposed.

Circular Dichroism↗
PubMed1991

The business planning process applied to an in-house corporate occupational medicine unit.

In using the business planning process we defined our services, determined the lowest available open market price for each, and multiplied the unit price by the number we performed in the year to calculate our income. All our costs were then allocated to these services, and a cost per service determined. These were compared to the commercially available rates. Our unit is the low cost provider by 10%, even when using worst case assumptions. We concluded that contracting with outside organizations for occupational medical services could not be justified on either a cost or a quality basis.

Competitive Bidding↗
PubMed1987

Immunologic response to the influenza virus neuraminidase is influenced by prior experience with the associated viral hemagglutinin. I. Studies in human vaccinees.

Analysis of an earlier study of H3N2 and H7N2 inactivated influenza vaccines in schoolchildren demonstrated a greater viral neuraminidase (NA) immunogenicity of the vaccine containing the H7 hemagglutinin (HA) antigen to which they had not been primed, despite the lesser NA antigen content of that vaccine. Thus, prior experience with the influenza viral HA appeared to have a negative influence on immune response to NA, the associated external glycoprotein, presumably on the basis of intermolecular antigenic competition. In a second study, sequential immunologic response to influenza viral NA was compared in college students who were immunized with either conventional commercial vaccine or an antigenic reassortant H7N1 vaccine, and who subsequently experienced natural infection with an H1N1 influenza virus. Although both vaccines were only marginally immunogenic in inducing NA antibody response in seronegative subjects, in vaccinees initially seropositive for HA antibody significant NA antibody titer increases occurred with H7N1 vaccine. Subsequent natural infection boosted NA antibody less effectively in the population previously primed by natural infection than in initially seronegative subjects primed by H7N1 vaccination. It is suggested that primary immunization monospecific for influenza viral NA may alter the subsequent pattern of immune response to one more favorable to the induction of NA antibody when virus is encountered.

Adult↗
PubMed1982

Mercury in takeaway fish in New Zealand.

Analysis of samples of takeaway fish cooked in batter collected in 1977 showed mercury concentrations in the fish flesh up to 4.4 micrograms/g; the highest concentrations were found in shark. The overall mean value found for the south Auckland areas was 0.72 microgram/g. At this level, the WHO provisional tolerable mercury intake for an adult would be supplied by approximately one mean (two pieces) per day of takeaway fish. Children eating similar sized meals would attain the tolerable mercury intake with one meal every second day.

Adult↗
PubMed1982

Effect of phalloidin on liver actin distribution, content, and turnover.

Phalloidin increases F-actin microfilament content and actin-directed immunofluorescence in hepatocytes in vivo and also increases actin polymerization and the stability of F-actin in vitro. We studied the sensitivity of immunofluorescent staining of actin to an actin depolymerizing factor (ADF) as well as actin content, degree of polymerization, and turnover in livers of in vivo phalloidin-treated rats. Pretreatment with ADF abolished anti-actin antibody (AAA) staining of normal liver but did not modify staining of livers from phalloidin-treated animals. Planimetric analyses of SDS-polyacrylamide gels showed the percent actin of total protein was increased by approximately 40% and the absolute amount of actin by approximately 43%, ten days after daily phalloidin treatment (50 micrograms/100 gm body weight). Similar but smaller changes could be seen after one day of treatment. Ultracentrifugational analyses of liver extracts indicated no change in the amount or proportion of G-actin but a 194% increase in the proportion of F-actin in ten-day treated animals, changes also apparent in one day animals. Neither the relative fractional rate of actin synthesis nor its synthesis as a percent of total protein synthesis was altered either at one-day or ten-day post-phalloidin treatment. Dualisotope experiments indicated that the rate of actin degradation was decreased selectively in the one- to three-day period following drug treatment. Thus, phalloidin appears to stabilize actin against the depolymerizing actions of ADF, increases the proportion of F-actin without altering the size of the G-actin pool, and causes accumulation of actin by decreasing its relative rate of degradation.

Actin Depolymerizing Factors↗
PubMed1980

Phosphorylation of heart glycogen synthase by cAMP-dependent protein kinase. Regulatory effects of ATP.

Glycogen synthase I, purified from bovine heart, had a specific activity of 33 units/mg and gave a single band on sodium dodecyl sulfate gel electrophoresis with a subunit molecular weight of 86,000. The enzyme was phosphorylated with cAMP-dependent protein kinase catalytic subunit, also isolated from heart. With 10 microM ATP, only one phosphate group was incorporated per subunit of glycogen synthase. The phosphorylation decreased the per cent of glycogen synthase I from 0.95 to 0.50 when activity was determined by assays with Na2SO4 and glucose 6-phosphate. Glycogen synthase containing one phosphate per subunit was designated GS-1. One additional phosphate was incorporated per synthase subunit when ATP was increased to 0.5 mM and the percent glycogen synthase I decreased from 0.50 to < 0.05. This enzyme form was designated GS-1,2. Conversion of GS-1 to Gs-1,2 gave cooperative kinetics with ATP concentration and a half-maximal stimulation at approximately 40 microM. Phosphorylation of GS-1 could also be achieved by adding other non-substrate nucleotide triphosphates such as ITP and UTP along with 10 microM ATP. Glucose-6-P and Na2SO4 were without effect on this phosphorylation reaction. Two separate peptides were obtained after CNBr cleavage of 32P-labeled GS-1,2 and only one from GS-1. Both enzyme forms contained a single phosphorylated peptide in common. Thus, heart glycogen synthase may be phosphorylated specifically in either of two different sites using appropriate concentrations of ATP. ATP acts as a substrate for the protein kinase and also affects the availability of a second site to phosphorylation by cAMP-dependent protein kinase.

Adenosine Triphosphate↗
PubMed1978

A ten year experience with cadaver kidney preservation using cryoprecipitated plasma.

Between August 1967 and January 1977, 699 cadaver kidneys were preserved and transplanted in our hospital after continuous perfusion with cryoprecipitated plasma. Overall graft survival of primary transplants was 55 +/- 2 per cent at one year and 41 +/- 2 per cent at four years. The results with ninety-six second transplants were similar. The number of HLA antigens shared and the duration of preservation did not influence graft survival. Patient survival among 426 cadaver graft recipients since September 1972, when lower dose immunosuppression was started, was 91 +/- 1 per cent at one year and 84 +/- 2 per cent at four years, significantly better than survival before then. Survival of fifty-two recipients of cadaver retransplants since September 1972 was 86 +/- 5 per cent at one year and 86 +/- 5 per cent at four years, which was better than before. The incidence of posttransplantation dialysis was 30 per cent and did not correlate with the length of preservation. Primary wound infections, primary ureteral extravasation, and vascular complications each occurred with an incidence of 1.1 per cent or less in patients treated with lower dose immunosuppression. Only four kidneys were lost because of complications, and in no instance was the need for transplant nephrectomy directly related to the method of preservation. Perfusion preservation with cryoprecipitated plasma gives excellent results compared with alternative methods.

Adolescent↗