A PCR-based method of screening individuals of all ages, from neonates to the elderly, for familial hyperaldosteronism type I

Aim: Unless specifically treated (glucocorticoids in low doses), Familial Hyperaldosteronism Type I(FH-I) may result in early death from stroke. We report the successful application of a rapid, polymerase chain reaction (PCR)-based method of detecting the 'hybrid' 11 beta-hydroxylase (11 beta-OHase)/aldosterone synthase (AS) gene as a screening test for FH-I. Methods: 'Long-PCR' was used to amplify, concurrently, a 4 kb fragment of AS gene (both primers AS-specific) and a 4 kb fragment of the hybrid gene (5' primer 11 beta-OHase-specific, 3'primer AS-specific) from DNA extracted from blood either collected locally or transported from elsewhere. Sample collection and transport were straightforward. This 4 kb fragment contains all the currently recognised hybrid gene 'crossover' points. Results: Within a single family, long-PCR identified all 21 individuals known to have FH-I. Hypertension was corrected in all 11 treated with glucocorticoids. Nine with normal blood pressure are being closely followed for development of hypertension. Long-PCR cord blood analysis excluded FH-I in three neonates born to affected individuals. Long-PCR newly identified two other affected families: (1) a female (60 years) with a personal and family history of stroke and her normotensive daughter (40 years), and (2) a female (51 years) previously treated for primary aldosteronism with amiloride, her two hypertensive sons (14 and 16 years) and her hypertensive mother (78 years). No false negative or false positive results have yet been encountered. At least seven other centres have successfully performed this test. Conclusion: Long-PCR is a reliable method of screening individuals of all ages for FH-I.

Improved methods for predicting individual leaf area and leaf senescence in maize (Zea mays)

The ability to predict leaf area and leaf area index is crucial in crop simulation models that predict crop growth and yield. Previous studies have shown existing methods of predicting leaf area to be inadequate when applied to a broad range of cultivars with different numbers of leaves. The objectives of the study were to (i) develop generalised methods of modelling individual and total plant leaf area, and leaf senescence, that do not require constants that are specific to environments and/or genotypes, (ii) re-examine the base, optimum, and maximum temperatures for calculation of thermal time for leaf senescence, and (iii) assess the method of calculation of individual leaf area from leaf length and leaf width in experimental work. Five cultivars of maize differing widely in maturity and adaptation were planted in October 1994 in south-eastern Queensland, and grown under non-limiting conditions of water and plant nutrient supplies. Additional data for maize plants with low total leaf number (12-17) grown at Katumani Research Centre, Kenya, were included to extend the range in the total leaf number per plant. The equation for the modified (slightly skewed) bell curve could be generalised for modelling individual leaf area, as all coefficients in it were related to total leaf number. Use of coefficients for individual genotypes can be avoided, and individual and total plant leaf area can be calculated from total leaf number. A single, logistic equation, relying on maximum plant leaf area and thermal time from emergence, was developed to predict leaf senescence. The base, optimum, and maximum temperatures for calculation of thermal time for leaf senescence were 8, 34, and 40 degrees C, and apply for the whole crop-cycle when used in modelling of leaf senescence. Thus, the modelling of leaf production and senescence is simplified, improved, and generalised. Consequently, the modelling of leaf area index (LAI) and variables that rely on LAI will be improved. For experimental purposes, we found that the calculation of leaf area from leaf length and leaf width remains appropriate, though the relationship differed slightly from previously published equations.

Improved one-step solid-phase extraction method for morphine, morphine-3-glucuronide, and morphine-6-glucuronide from plasma and quantitation using high-performance liquid chromatography with electrochemical detection

This communication describes an improved one-step solid-phase extraction method for the recovery of morphine (M), morphine-3-glucuronide (M3G), and morphine-6-glucuronide (M6G) from human plasma with reduced coextraction of endogenous plasma constituents, compared to that of the authors' previously reported method. The magnitude of the peak caused by endogenous plasma components in the chromatogram that eluted immediately before the retention time of M3G has been reduced (similar to 80%) significantly (p < 0.01) while achieving high extraction efficiencies for the compounds of interest, viz morphine, M6G, and M3G (93.8 +/- 2.5, 91.7 +/- 1.7, and 93.1 +/- 2.2%, respectively). Furthermore, when the improved solid-phase extraction method was used, the extraction cartridge-derived late-eluting peak (retention time 90 to 100 minutes) reported in our previous method, was no longer present in the plasma extracts. Therefore the combined effect of reducing the recovery of the endogenous components of plasma that chromatographed just before the retention time of M3G and the removal of the late-eluting, extraction cartridge-derived peak has resulted in a decrease in the chromatographic run-time to 20 minutes, thereby increasing the sample throughput by up to 100%.

Electrical impedance particle size method (Coulter Counter) detects the large fat globules in poorly homogenised UHT processed milk

The large fat globules that can be present in UHT milk due to inadequate homogenisation cause a cream layer to form that limits the shelf life of UHT milk. Four different particle size measurement techniques were used to measure the size of fat globules in poorly homogenised UHT milk processed in a UHT pilot plant. The thickness of the cream layer that formed during storage was negatively correlated with homogenisation pressure. It was positively correlated with the mass mean diameter and the percentage volume of particles between 1.5 and 2 mu m diameter, as determined by laser light scattering using the Malvern Mastersizer. Also, the thickness of the cream layer was positively correlated with the volume mode diameter and the percentage volume of particles between 1.5 and 2 mu m diameter, as determined by electrical impedance using the Coulter Counter. The cream layer thickness did not correlate significantly with the Coulter Counter measurements of volume mean diameter, or volume percentages of particles between 2 and 5 mu m or 5 and 10 mu m diameter. Spectroturbidimetry (Emulsion Quality Analyser) and light microscopy analyses were found to be unsuitable for assessing the size of the fat particles. This study suggests that the fat globule size distribution as determined by the electrical impedance method (Coulter Counter) is the most useful for determining the efficiency of homogenisation and therefore for predicting the stability of the fat emulsion in UHT milk during storage.

A study of the stability of subcycling algorithms in structural dynamics

Algorithms for explicit integration of structural dynamics problems with multiple time steps (subcycling) are investigated. Only one such algorithm, due to Smolinski and Sleith has proved to be stable in a classical sense. A simplified version of this algorithm that retains its stability is presented. However, as with the original version, it can be shown to sacrifice accuracy to achieve stability. Another algorithm in use is shown to be only statistically stable, in that a probability of stability can be assigned if appropriate time step limits are observed. This probability improves rapidly with the number of degrees of freedom in a finite element model. The stability problems are shown to be a property of the central difference method itself, which is modified to give the subcycling algorithm. A related problem is shown to arise when a constraint equation in time is introduced into a time-continuous space-time finite element model. (C) 1998 Elsevier Science S.A.

Speciation of arsenic metabolites in the urine of occupational workers and experimental rats using an optimised hydride cold-trapping method

A hydride cold-trapping technique was developed and optimised for the measurement of urinary arsenic metabolites. The analytical precision of the method was found to be 6.1, 4.0 and 4.8% (n = 5) for inorganic arsenic (As-i), monomethylarsonate (MMA) and dimethylarsinate (DMA), respectively, with recoveries close to 100%, The detection limits were 1.0, 1.3 and 3 ng for As-i, MMA and DMA, respectively. The method was then used to analyse urine samples obtained from three groups of workers for occupational exposure in three companies where copper chrome arsenate was used for timber treatment. The results were compared with those for a normal control group of laboratory workers. Arsenic and its metabolites were also measured in experimental rats given 5 mg As kg(-1) body mass by oral gavage in the form of sodium arsenite, calcium arsenite or sodium arsenate. Occupational workers showed a significantly higher excretion of As-i, Up to two fold increases of urinary As-i excretion in rats compared with control rats were also observed in animals dosed with various forms of arsenicals. The method is suitable for the measurement of arsenic metabolites in urine of both humans and experimental animals.

Solid-phase extraction method with high-performance liquid chromatography and electrochemical detection for the quantitative analysis of oxycodone in human plasma

A sensitive and reproducible solid-phase extraction (SPE) method for the quantification of oxycodone in human plasma was developed. Varian Certify SPE cartridges containing both C-8 and benzoic acid functional groups were the most suitable for the extraction of oxycodone and codeine (internal standard), with consistently high (greater than or equal to 80%) and reproducible recoveries. The elution mobile phase consisted of 1.2 ml of butyl chloride-isopropanol (80:20, v/v) containing 2% ammonia. The quantification limit for oxycodone was 5.3 pmol on-column. Within-day and inter-day coefficients of variation were 1.2% and 6.8% respectively for 284 nM oxycodone and 9.5% and 6.2% respectively for 28.4 nM oxycodone using 0.5-ml plasma aliquots. (C) 1998 Elsevier Science BN. All rights reserved.

The relationship between plasma lactate parameters, W-peak and 1-h cycling performance in women

Purpose: The relationship between six descriptors of lactate increase, peak (V) over dot O-2,W-peak, and 1-h cycling performance were compared in 24 trained, female cyclists (peak (V) over dot O-2 = 48.11 +/- 6.32 mL . kg(-1) . min(-1)). Methods: The six descriptors of lactate increase were: 1) lactate threshold (LT; the power output at which plasma lactate concentration begins to increase above the resting level during an incremental exercise test), 2) LT1 (the power output at which plasma lactate increases by 1 mM or more), 3) LTD (the lactate threshold calculated by the D-max method), 4) LTMOD (the lactate threshold calculated by a modified D-max method), 5) L4 (the power output at which plasma lactate reaches a concentration of 4 mmol-L-1), and 6) LTLOG (the power output at which plasma lactate concentration begins to increase when the log([La-]) is plotted against the log (power output)). Subjects first completed a peak (V) over dot O-2 test on a cycle ergometer. Finger-tip capillary blood was sampled within 30 s of the end of each 3-min stage for analysis of plasma lactate. Endurance performance was assessed 7 d later using a 1-h cycle test (OHT) in which subjects were directed to achieve the highest possible average power output. Results: The mean power output (W) for the OHT (+/- SD) was 183.01 +/- 18.88, and for each lactate variable was: LT (138.54 +/- 46.61), LT1 (179.17 +/- 27.25), LTLOG (143.97 +/- 45.74), L4 (198.09 +/- 33.84), LTD (178.79 +/- 24.07), LTMOD (212.28 +/- 31.75). Average power output during the OHT was more strongly correlated with all plasma lactate parameters (0.61 < r < 0.84) and W-peak (r = 0.81) than with peak (V) over dot O-2 (r = 0.55). The six lactate parameters were strongly correlated with each other (0.54 < r < 0.91) and of the six lactate parameters, LTD correlated best with endurance performance (r = 0.84). Conclusions: It was concluded that plasma lactate parameters and W-peak provide better indices of endurance performance than peak (V) over dot O-2 and that, of the six descriptors of lactate increase measured in this study, LTD is most strongly related to 1-h cycling performance in trained, female cyclists.

Simultaneous detection of multiple antigens with triple immunolabeling

Immunolabeling is commonly used to localize antigens within frozen or paraffin tissue sections. We modified existing immunolabeling techniques to allow the detection of three antigens simultaneously within the one tissue section. The approach relies on the use of three monoclonal antibodies in sequential immunoperoxidase staining steps, each with colored substrates, resulting in the deposition of black, brown, and rose stains. The method is rapid and does not require novel techniques or materials. In this report, we demonstrate the colocalization of mast cell tryptase, neurofilament protein, and CD31 (platelet-endothelial cell adhesion molecule) or laminin in normal human skin and normal buccal mucosa, as an illustration of the power and simplicity of the multiple antigen localization technique.

The Modified Card Sorting Test: Test-retest stability and relationships with demographic variables in a healthy older adult sample

Objectives. To investigate the test-retest stability of a standardized version of Nelson's (1976) Modified Card Sorting Test (MCST) and its relationships with demographic variables in a sample of healthy older adults. Design. A standard card order and administration were devised for the MCST and administered to participants at an initial assessment, and again at a second session conducted a minimum of six months later in order to examine its test-retest stability. Participants were also administered the WAIS-R at initial assessment in order to provide a measure of psychometric intelligence. Methods. Thirty-six (24 female, 12 male) healthy older adults aged 52 to 77 years with mean education 12.42 years (SD = 3.53) completed the MCST on two occasions approximately 7.5 months (SD = 1.61) apart. Stability coefficients and test-retest differences were calculated for the range of scores. The effect of gender on MCST performance was examined. Correlations between MCST scores and age, education and WAIS-R IQs were also determined. Results. Stability coefficients ranged from .26 for the percent perseverative errors measure to .49 for the failure to maintain set measure. Several measures were significantly correlated with age, education and WAIS-R IQs, although no effect of gender on MCST performance was found. Conclusions. None of the stability coefficients reached the level required for clinical decision making. The results indicate that participants' age, education, and intelligence need to be considered when interpreting MCST performance. Normative studies of MCST performance as well as further studies with patients with executive dysfunction are needed.

Comparison of carbohydrate-deficient transferrin, immunoglobulin A antibodies reactive with acetaldehyde-modified protein and acetaldehyde-modified albumin with conventional markers of alcohol consumption

Carbohydrate-deficient transferrin (CDT) has emerged as the best new marker for alcohol abuse. Recently plasma immunoglobulin A (IgA) reactivity with acetaldehyde (AcH)-modified proteins, or the modified proteins per se, have been proposed as a markers for high levels of alcohol consumption. In this study, we have compared CDT, IgA reactivity with AcH adducts (IgA ASR), and AcH-modified albumin with conventional markers of high alcohol intake in groups with well-defined drinking histories, The plasma activity of ALT, AST, and gamma-glutamyltransferase increased steadily with increasing alcohol consumption, CDT and AcH-modified albumin showed a similar pattern, whereas IgA ASR appeared only to be elevated after a threshold level of consumption had been reached, Neither CDT IgA ASR or AcH-modified albumin correlated strongly with any of the conventional markers or each other. This study shows that CDT, IgA ASR, AcH-modified albumin, and the conventional markers are not related, but suggests that the concurrent use of CDT and IgA ASR may lead to better identification of high alcohol intake.

A reproducible method for automated extraction of brain volumes from 3D human head MR images

An automated method for extracting brain volumes from three commonly acquired three-dimensional (3D) MR images (proton density, T1 weighted, and T2-weighted) of the human head is described. The procedure is divided into four levels: preprocessing, segmentation, scalp removal, and postprocessing. A user-provided reference point is the sole operator-dependent input required, The method's parameters were first optimized and then fixed and applied to 30 repeat data sets from 15 normal older adult subjects to investigate its reproducibility. Percent differences between total brain volumes (TBVs) for the subjects' repeated data sets ranged from .5% to 2.2%. We conclude that the method is both robust and reproducible and has the potential for wide application.

A novel method for tailoring the pore-opening size of MCM-41 materials

A novel pore tailoring method is proposed by which the pore-opening sizes of MCM-41 materials can be finely tuned without significant loss in pore volume and surface area.

A method of analysing stream channel response to environmental change: gauge data for the Tully River

Gauging data are available from numerous streams throughout Australia, and these data provide a basis for historical analysis of geomorphic change in stream channels in response to both natural phenomena and human activities. We present a simple method for analysis of these data, and a briefcase study of an application to channel change in the Tully River, in the humid tropics of north Queensland. The analysis suggests that this channel has narrowed and deepened, rather than aggraded: channel aggradation was expected, given the intensification of land use in the catchment, upstream of the gauging station. Limitations of the method relate to the time periods over which stream gauging occurred; the spatial patterns of stream gauging sites; the quality and consistency of data collection; and the availability of concurrent land-use histories on which to base the interpretation of the channel changes.

Protein sequence threading, the alignment problem, and a two-step strategy

Conventionally, protein structure prediction via threading relies on some nonoptimal method to align a protein sequence to each member of a library of known structures. We show how a score function (force field) can be modified so as to allow the direct application of a dynamic programming algorithm to the problem. This involves an approximation whose damage can be minimized by an optimization process during score function parameter determination. The method is compared to sequence to structure alignments using a more conventional pair-wise score function and the frozen approximation. The new method produces results comparable to the frozen approximation, but is faster and has fewer adjustable parameters. It is also free of memory of the template's original amino acid sequence, and does not suffer from a problem of nonconvergence, which can be shown to occur with the frozen approximation. Alignments generated by the simplified score function can then be ranked using a second score function with the approximations removed. (C) 1999 John Wiley & Sons, Inc.