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Biomedical subjects

W G Miller

Publications and source records attributed to W G Miller.

At least 55 records · Page 3Linked to original sources
PubMed1987

Escherichia coli tryptophan repressor binds multiple sites within the aroH and trp operators.

DNase I footprinting and methylation protection studies have been used to analyze the binding of Escherichia coli Trp repressor to the trpR, aroH, and trp operators. The methylation protection assay shows that Trp repressor binds in two successive major grooves of the trpR operator, three successive major grooves of the aroH operator, and four successive major grooves of the trp operator. The simplest model that explains the difference in Trp repressor interaction at the three operators is that the aroH and trp operators are composed of multiple, helically stacked binding sites. When viewed in three dimensions, each site is positioned on a different face of the DNA, and together process up the surface of the DNA helix. Analysis of a deletion derivative of the trp operator supports this model.

Bacterial Proteins↗
PubMed1987

Absorbance errors from using pieced-together rotors in the Roche Cobas-Bio centrifugal analyzer.

Two Cobas-Bio analyzers were found to have significantly increased imprecision, owing to absorbance errors, when we used rotors pieced together from segments of four to 10 cuvets. The difference in CV between whole/unbroken and four-cuvet or 10-cuvet segment rotors was significant by Student's unpaired t-test (P = 0.038 and less than 0.0001, respectively) and by the Mann-Whitney U test (P less than 0.0001 for either). Pieced-together rotors were found to have plastic fragments in some cuvets, both adjacent to the break points and in other positions within the segment. There was no significant difference in delta A between adjacent cuvets at break positions or in between break positions by unpaired t-test (P = 0.214) or by the Mann-Whitney U test (P = 0.235). There was a significant difference by either test (P less than 0.0001) for the delta A between adjacent cuvets in broken rotors and those in whole/unbroken rotors. We conclude that random errors in absorbance are observed both in cuvets adjacent to break positions and in cuvets removed from the break point when pieced-together rotors are used.

Autoanalysis↗
PubMed1987

Optical kinetic method for calibration of spectrophotometer temperature: demonstration with the Cobas-Bio analyzer.

The pseudo-first-order rate constant for the Jaffé reaction with creatinine varies logarithmically with temperature and was calibrated in the range 25 to 37 degrees C to measure the temperature of the liquid in the lightpath of spectrophotometric instrumentation. The reagent concentrations can be adjusted to permit rate-constant measurements in time intervals from a few seconds to several minutes. The temperature increment that can be resolved is limited only by the analytical imprecision of the instrumentation used to measure the rate constant and the calibration temperature. In this investigation, a temperature SD of 0.03 degrees C could be measured. Two Cobas-Bio centrifugal analyzers, used to demonstrate the utility of this technique, were found to have temperature errors from -1.0 to -1.7 degrees C in the 30 to 37 degrees C range and overall temperature SD of 0.19 and 0.36 degree C, respectively, at 37 degrees C. Analysis of variance gave between-rotor SD of 0.14 and 0.34 degrees C and within-rotor SD of 0.13 and 0.11 degree C, respectively. We found temperature differences of 0.3 degree C between cuvets in a rotor, and gradients of 0.3 and 0.4 degree C, respectively, from the top to bottom of an individual cuvet in the two instruments.

Analysis of Variance↗
PubMed1986

Interlaboratory standardization of enzyme results: the Richmond project.

Three concentrations of proficiency monitoring material and two concentrations of secondary standard calibrating material were prepared and stored frozen. The materials were prepared in buffer containing amylase from human saliva, aspartate aminotransferase from human liver, creatine kinase from human muscle, human serum albumin (20 g/L), and cofactors. The proficiency monitoring material was assayed by 10 methods in nine laboratories for 15 days to establish baseline performance. Each laboratory then used the secondary standard calibrating material to calibrate their instruments' responses to that of a standardization method, and repeated the assay of the proficiency monitoring material for 15 days. For amylase before calibration, between-laboratory mean values for the three concentrations of proficiency monitoring material were 29% lower than the standardization method, and the between-laboratory CV was 28%. After calibration the mean amylase values were 4% lower and the CV was 6%. For aspartate aminotransferase, the pre-calibration between-laboratory mean values were 24% higher than the standardization method (CV 14%) but only 3% higher (CV 6%) after calibration. CK activity deteriorated at storage temperatures above -70 degrees C. This study demonstrates that, by using a common secondary standard, laboratories can improve calibration of enzyme results.

Amylases↗
PubMed1986

Monitoring the stability of wavelength calibration of spectrophotometers.

The difference in absorbance (delta A) between equimolar acid and alkaline solutions of methyl red, at a wavelength near the isosbestic point of the indicator, is reproducible. Furthermore, this delta A is sensitive to changes in the wavelength calibration of the instrument used to make the measurement. The delta A of methyl red can be used to monitor wavelength accuracy in both manual and automated spectrophotometric instruments. Although this measurement does not establish wavelength calibration, it is useful for monitoring the wavelength accuracy of previously calibrated, automated spectrophotometers that do not easily lend themselves to calibration checks by conventional techniques.

Azo Compounds↗
PubMed1985

Cemental annulation enhancement: a technique for age determination in man.

Cemental annulations are easily countable in teeth from animals that have an exaggerated regular change of food intake from season to season. Although present in human teeth, cemental annulations are not always easy to count. A method for preparing human teeth for evaluation involving collection, identification, measuring, sectioning, cleaning, acid etching, staining, and mounting is reported. Sections 100-microns thick were stained with cresyl fast violet as a stain of choice and were photographed using standard light microscopic techniques as well as Nomarsky interference microscopy. Countability of annulations from photographic enlargements was evaluated.

Aging↗
PubMed1985

Prediction of carrier status in Duchenne dystrophy by creatine kinase measurement.

Serum creatine kinase (CK) was measured in 515 healthy white women and 28 obligate carriers for Duchenne muscular dystrophy. There was substantial overlap between the control and carrier populations. To analyze the impact of the degree of overlap, the predictive value of a CK result was determined by (1) using sensitivity and specificity analysis, which assumes dichotomization into a positive or negative result based on a particular cut-off value; and (2) using likelihood ratio analysis, which evaluates an individual result based on the continuum observed for control and carrier populations. There was no clinically important difference whether an observed 57% or a hypothetical 33% overlap between control and carrier results was used. Because of the substantial overlap, the CK test utility is limited to those suspected carriers whose results fall above the healthy population interval. A low CK result does not provide sufficient assurance of noncarrier status.

Adult↗
PubMed1984

Blind evaluation of lymphocyte capping in Duchenne muscular dystrophy.

Recently, Pickard et al reported decreased "capping" in lymphocytes from patients with Duchenne type muscular dystrophy (DMD) as well as female carriers of the DMD trait. To resolve subsequent debate about the reproducibility of this finding, we carried out a "blinded" collaborative study designed to eliminate the possibility of observer bias. Blood samples from DMD patients, their mothers, and controls were obtained and coded at Johns Hopkins and transported to the Medical College of Virginia, where lymphocyte capping was tested using FITC-labeled polyvalent anti-human immunoglobulin. Diminished capping in lymphocytes was found in 12 of 13 DMD patients (17 of 18 blood samples) and in 14 of 17 mothers of DMD patients (19 of 23 blood samples), as compared with 8 of 21 control subjects (8 of 22 blood samples). The results in both the patient and the carrier groups differed significantly from those in the control group, confirming previous observations of diminished lymphocyte capping in DMD. The findings provide support for the concept of a systemic defect associated with cell membranes in this disorder. The relatively high incidence of false positive results limits the usefulness of lymphocyte capping as a diagnostic test for carriers under the conditions of this study.

Female↗
PubMed1984

Sampling from a skewed population distribution as exemplified by estimation of the creatine kinase upper reference limit.

Creatine kinase (EC 2.7.3.2) was measured in sera from 580 females, ages 1-77 years, and 550 males, ages 1-63 years. The distribution of results for male and female groups shows pronounced skewing toward higher values. The observed distribution of results could not be described by any of six mathematical formulas for skewed distributions, an indication of the unsuitability of such formulas to transform these data for parametric analysis. The range of 97.5 percentile estimates produced by six independent samples of 100, 200, and 400 observations randomly selected from a mathematical model defined by the adult female distribution showed progressive narrowing from the 150-380 U/L interval for the samples of 100 observations to 200-265 U/L for the samples of 400 observations; no further improvement was seen when 800 observations were used. The samples of 100 and 200 observations contained extreme value points that might appear as "outliers" but were shown to be valid members of the population distribution when larger sample sizes were collected.

Adolescent↗
PubMed1984

Are reference limits for serum creatine kinase valid in detection of the carrier state for Duchenne muscular dystrophy?

We evaluated serum creatine kinase (CK) as an index to heterozygosity in Duchenne muscular dystrophy. When the 97.5th percentile of the CK normal reference interval was selected as the cutoff point, only 31% of 28 obligate carrier mothers and 24% of 43 mothers of simplex cases (those with only one occurrence of dystrophy in the kindred) exceeded this cutoff value. The outcome depended to some degree on the method used for establishing the reference limit for 379 presumably non-carrier, ambulatory women. The considerable overlap of CK activities between the control and carrier population as well as the non-gaussian distribution of the data permitted no satisfactory approach for differentiating these two populations. Neither the application of likelihood ratios, which evaluates a continuum of results without the dichotomy of a cutoff point, nor the application of predictive value based on sensitivity and specificity, which involves use of a cutoff value for decision making, provided a reliable estimate of carrier status. There was no significant difference (2 alpha = .19) between the median CK activity of obligate carrier mothers and mothers of simplex cases. The serum CK test does not provide data that either support or reject the Haldane hypothesis.

Adolescent↗
PubMed1983

The temperature control system of the Cobas-Bio centrifugal analyzer.

The temperature control system of the Cobas-Bio centrifugal analyzer (Roche Analytical Instruments, Montclair, NJ) was evaluated using a calibrated thermistor probe and a solution of cresol red in tris buffer as an "optical thermometer" to measure the temperature of the cuvette contents during operation. The instrument accurately achieves a set temperature of 30 degrees C or 37 degrees C, within thermistor readability of 0.13 degrees C, and maintains that temperature within +/- 0.05 to 0.08 degrees C for at least 18 minutes. There is negligible temperature difference between cuvettes in a rotor during operation. After pipetting samples and reagents into the rotor, 1.5 minutes at 30 degrees C and 2 minutes at 37 degrees C are required to bring the cuvette contents within 0.1 degree C of the set temperature. If a second reagent representing 10% of the total volume is added to cuvettes that have been preincubated at the set temperature, up to 1.5 minutes at 30 degrees C and 2 minutes at 37 degrees C are required to reach temperature equilibrium.

Centrifugation↗
PubMed1982

A calibration protocol for serum-based secondary standards.

In this procedure, tentative calibration values are assigned to secondary standards by analysis of that material with the routinely used instrument calibrated by use of the former lot of material. Aliquots of serum from patients are then analyzed by the routine method and an appropriate calibration method in which primary standards are used. If the patient-sera results by both methods are the same, the assigned calibration value is assumed to be correct; if not, the calibration value of the secondary-standard material is adjusted to produce agreement in patient-sera results between the two methods. Thus, calibration of the routine method is linked via the calibration method to primary standards as the criterion of accuracy. This approach corrects for matrix interference of serum-based secondary standards. All that is expected for the secondary standard to obtain an accurate result for patient sera is to produce an analytical signal (irrespective of the origin of the signal) that is equivalent to the calibration value. Differences were significant between the secondary-standard calibration value we determined by this procedure and that assigned by the manufacturer for bilirubin, calcium, phosphorus, chloride, CO2, creatinine, glucose, sodium, uric acid, and commonly measured enzymes.

Autoanalysis↗
PubMed1981

Network structure in gels of rod-like polypeptides.

The formation of a mechanically self-supporting, macromolecular gel or network is understood on a molecular scale in terms of cross-links or branch points, either of a permanent nature by covalent bond formation, or of a reversible nature, such as in the gelation of gelatin. In thermally reversible gels each branch point may contain numerous monomeric units, indicative of local crystallite or aggregate formation, as in cellulose acetate or gelatin, or only two monomeric units as in polyacrylylglycinamide. The formation of reversible networks in nonionic, rod-like polypeptide homopolymers, particularly at low concentrations (<0.1 wt%), is surprising in that the molecular origin of the branch points is not obvious. We have suggested previously that this network constitutes a thermodynamic phase resulting from a particular kinetic mechanism of phase formation made favourable by the unusual polymerdiluent phase equilibria occurring with stiff-chain polymers. Here, the network is visualized by electron microscopy, and shown to be compatible with the proposed mechanism.

Biopolymers↗
PubMed1976

On the design of clinical chemistry quality-control sera.

Quality-control specimens having quantitave interspecimen relationships were prepared by (a) making admixtures of two serum pools and (b) making various dilutions of the admixtures. The specimens were analyzed for many constituents to illustrate how the data obtained could be manipulated and used. The inter-related specimens permit the reduction of quality-control data to a few target values and should be particularly useful for the evaluation of multi-channel analyzers, and for use in inter-laboratory proficiency surveys. Aspects of the design of quality control and proficiency test specimens are also discussed.

Alkaline Phosphatase↗
PubMed1975

Electron transport by C-type cytochromes. I. The reaction of horse heart cytochrome c with anionic reductants.

The kinetics of reduction of horse heartcytochrome c have been investigated using the reductants sodium dithionite and potassium ferrocyanide. Sodium dithionite reduction at pH 7.0 yields rate constants of 2.8 X 10(8)M(-1)sec-1 for SO2 AND 6 X 10(5) M-1 sec-1 for S2O4 at infinite dilution. Moreover, the data presented demonstrates the participation of positively charged amino acid side chains at the site of electron transfer. The effect of pH on the reduction of ferricytochrome c requires a minimum of two pK values for description (pK1 = 7.0 +/- 0.4, pK2 = 9.3 +/- 0.3). Based on the pK values determined, one or more lysines and a residues(s) with a low pK are implicated as the positively charged residues participating in electron transfer. From a comparison of the rates of reduction of various denatured forms of cytochrome c we feel that the most viable conclusion is that electron transfer takes place at the exposed heme edge in the vicinity of the amino acid side chains indicated above. Ferrocyanide reduction of ferri-horse heart cytochrome c takes place in a kinetically complex manner. A mechanism is described which includes complexes of ferrocyanide and ferricytochrome c and ferricyanide and ferrocytochrome c. As was found for dithionite reduction a positively charged region of the cytochrome c participates in electron transfer. Combining our results with ferrocyanide and dithionite we conclude that avaible data is compatible with a single mechanism of electron transfer. It is suggested that the kinetic distinction between different reductants lies in the lifetime of the transient complex formed, with the order ferrocyanide greater than S2O4 greater than SO2.

Cacodylic Acid↗