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Biomedical subjects

J Payne

Publications and source records attributed to J Payne.

At least 19 recordsLinked to original sources
PubMed2001

Identification, purification, and characterization of iminodiacetate oxidase from the EDTA-degrading bacterium BNC1.

Microbial degradation of synthetic chelating agents, such as EDTA and nitrilotriacetate (NTA), may help immobilizing radionuclides and heavy metals in the environment. The EDTA- and NTA-degrading bacterium BNC1 uses EDTA monooxygenase to oxidize NTA to iminodiacetate (IDA) and EDTA to ethylenediaminediacetate (EDDA). IDA- and EDDA-degrading enzymes have not been purified and characterized to date. In this report, an IDA oxidase was purified to apparent homogeneity from strain BNC1 by using a combination of eight purification steps. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis revealed a single protein band of 40 kDa, and by using size exclusion chromatography, we estimated the native enzyme to be a homodimer. Flavin adenine dinucleotide was determined as its prosthetic group. The purified enzyme oxidized IDA to glycine and glyoxylate with the consumption of O2. The temperature and pH optima for IDA oxidation were 35 degrees C and 8, respectively. The apparent Km for IDA was 4.0 mM with a kcat of 5.3 s(-1). When the N-terminal amino acid sequence was determined, it matched exactly with that encoded by a previously sequenced hypothetical oxidase gene of BNC1. The gene was expressed in Escherichia coli, and the gene product as a C-terminal fusion with a His tag was purified by a one-step nickel affinity chromatography. The purified fusion protein had essentially the same enzymatic activity and properties as the native IDA oxidase. IDA oxidase also oxidized EDDA to ethylenediamine and glyoxylate. Thus, IDA oxidase is likely the second enzyme in both NTA and EDTA degradation pathways in strain BNC1.

Amino Acid Sequence↗
PubMed2001

Mixtures of four organochlorines enhance human breast cancer cell proliferation.

In view of the large differences between the concentrations of estrogenic chemicals needed to elicit effects in in vitro assays and their levels in human tissues, it is hard to explain possible health risks in terms of exposure to individual compounds. Human populations, however, are exposed to mixtures of estrogenic and estrogen-like agents and it is necessary to consider the impact of combined effects. We assessed the combined effects of 1-(o-chlorophenyl)-1-(p-chlorophenyl)-2,2,2-trichloroethane (o,p'-DDT), 2,2-bis(p-chlorophenyl)-1,1-dichloroethylene (p,p'-DDE), beta-hexachlorocyclohexane (beta-HCH), and 1,1-bis(p-chlorophenyl)-2,2,2-trichloroethane (p,p'-DDT) on the induction of cell proliferation in MCF-7 cells. All four compounds are persistent organochlorines that can be found in human tissues. We performed extensive concentration-response analyses with the single agents to predict the effects of two mixtures of all four compounds with different mixture ratios. We calculated the predictions by using the pharmacologically well-founded models of concentration addition and independent action and then tested them experimentally. o,p'-DDT, p,p'-DDE, beta-HCH, and p,p'-DDT acted together to produce proliferative effects in MCF-7 cells. The combined effect of the four agents could be predicted on the basis of data about single agent concentration-response relationships. Regression analysis demonstrated that there were combination effects even when each mixture component was present at levels at or below its individual no-observed-effect-concentration. We assessed combination effects in two ways: First, evaluations in relation to the proliferative responses induced by single mixture components revealed that the combination effects were stronger than the effects of the most potent constituent. Thus, according to this method of evaluation, the combined effects may be termed synergistic. Second, comparisons with the expected effects, as predicted by concentration addition and independent action, showed excellent agreement between prediction and observation. With this approach, the combined effect of all four compounds can be termed additive.

Breast Neoplasms↗
PubMed2000

Improving the reproducibility of the MCF-7 cell proliferation assay for the detection of xenoestrogens.

The MCF-7 cell proliferation assay is potentially a simple and highly reproducible tool for the identification of estrogenic compounds. However, its widespread use has been complicated by the lack of a standardised protocol, resulting in considerable inter-laboratory variability. We have explored the sources of variability both in relation to cell lines and test regimens and report on optimised procedures for the identification of estrogenic agents. Two supposedly identical MCF-7 parent cell lines (designated UCL and SOP), and the BUS subline were cultured according to an existing protocol, and responses to 17-estradiol (E2) assessed. Despite yielding almost identical EC50 values, the proliferative response varied widely between cell lines from 0.98-fold over controls (UCL) to 8.9-fold (BUS) indicating major differences between them. The underlying causes may be genetic, and to assess this we used comparative genomic hybridisation (CGH), a technique which allows the detection of DNA sequence copy number changes on a genome-wide scale. Although numerous similarities existed between the different cell lines, the least oestrogen-responsive line (MCF-7/UCL) exhibited the greatest number of cytogenetic changes, many of which were not seen in MCF-7/SOP cells. We suggest that care must be taken, therefore, when choosing a cell line for MCF-7 cell-based experiments. Selecting the MCF-7/SOP line for further work, we carried out a thorough and systematic optimisation of the MCF-7 cell proliferation assay, finding that a 72-h period in oestrogen-free medium before treatment strongly influenced the cells response to E2. With 1 nM E2, proliferation increased from 1.5-fold to 6.5-fold relative to vehicle-treated controls, a response similar to that seen with MCF-7/BUS cells in the E-SCREEN protocol devised by Soto et al. With parent MCF-7 cells, other laboratories have reported only 4.5-fold increases as maximal. Here we present evidence that the choice of cell line and culture conditions are crucial in determining test outcomes, and once chosen and adhered to the assay yields reproducible results.

Cell Division↗
PubMed2000

Calcium signaling induced by adhesion mediates protein tyrosine phosphorylation and is independent of pHi.

Our goal was to evaluate early signaling events that occur as epithelial cells make initial contact with a substrate and to correlate them with phosphorylation. The corneal epithelium was chosen to study signaling events that occur with adhesion because it represents a simple system in which the tissue adheres to a basal lamina, is avascular, and is bathed by a tear film in which changes in the local environment are hypothesized to alter signaling. To perform these experiments we developed a novel adhesion assay to capture the changes in intracellular Ca(2+) and pH that occur as a cell makes its initial contact with a substrate. The first transient cytosolic Ca(2+) peak was detected only as the cell made contact with the substrate and was demonstrated using fluorimetric assays combined with live cell imaging. We demonstrated that this transient Ca(2+) peak always preceded a cytoplasmic alkalization. When the intracellular environment was modified, the initial response was altered. Pretreatment with 1,2-bis(o-aminophenoxy)ethane-N,N, N'N'-tetraacetic acid (BAPTA), an intracellular chelator, inhibited Ca(2+) mobilization, whereas benzamil altered the duration of the oscillations. Thapsigargin caused an initial Ca(2+) release followed by a long attenuated response. An inositol triphosphate analog induced a large initial response, whereas heparin inhibited Ca(2+) oscillations. Inhibitors of tyrosine phosphorylation did not alter the initial mobilization of cytosolic Ca(2) but clearance of cytosolic Ca(2+) was inhibited. Exposing corneal epithelial cells to BAPTA, benzamil, or thapsigargin also attenuated the phosphorylation of the focal adhesion protein paxillin. However, although heparin inhibited Ca(2+) oscillations, it did not alter phosphorylation of paxillin. These studies demonstrate that the initial contact that a cell makes with a substrate modulates the intracellular environment, and that changes in Ca(2+) mobilization can alter later signaling events such as the phosphorylation of specific adhesion proteins. These findings may have implications for wound repair and development.

Animals↗
PubMed2000

Iron ore mines leachate potential for oxyradical production.

The ecotoxicological effects of mining effluents is coming under much greater scrutiny. It appears necessary to explore possible health effects in association with iron ore mining effluents. The present results clearly demonstrate that iron-ore leachate is not an inert media but has the potential to induce lipid peroxidation. Peroxidation was assessed by measuring oxygen consumption in the presence of a reducing agent such as ascorbate or NADPH and a chelator such as EDTA. Labrador iron ore is an insoluble complex crystalline material containing a mixture of metals (Fe, Al, Ti, Mn, Mg,ellipsis, ) in contrast to the iron sources used for normal lipid peroxidation studies. The metal of highest percentage is iron (59. 58%), a metal known to induce oxyradical production. Iron ore powder initiated ascorbic acid-dependent lipid peroxidation (nonenzymatic) in liposomes, lipids extracted from rat and salmon liver microsomes, and intact salmon liver microsomes. It also revealed an inhibitory effect of NADPH-dependent microsomes lipid peroxidation as well as on NADPH cytochrome c reductase activity. However, nonenzymatic peroxidation in rat liver microsomes was not significantly inhibited. Cytochrome P450 IA1- and IIB1-dependent enzymatic activities as well as P450 levels were not affected. The inhibition could be due to one of the other components of iron ore leachate (Mn, Al,ellipsis, ). These effects of iron-ore leachate indicate that a potential toxicity could be associated with its release into lakes. Further studies are necessary to explore in vivo effects on aquatic animals.

Animals↗
PubMed2000

Tyrosine phosphorylation: a critical component in the formation of hemidesmosomes.

Our goal was to evaluate the role of tyrosine phosphorylation in the complete formation of hemidesmosomes that occurs during development or during remodeling after injury. A corneal organ culture system was used to study hemidesmosome formation as it would occur in an intact tissue. Phosphorylation of the integrin subunit beta 4 and bullous pemphigoid antigen-1 (BPAG-1) was examined, as these proteins are known to play a role in linking the electron-dense plaques along the basal surface with the intermediate filaments to complete the formation of hemidesmosomes. Corneal epithelial sheets were placed on substrata that contained an intact basal lamina or basal laminae that had been either modified or removed. These constructs were incubated for up to 18 h, and hemidesmosome formation was evaluated by using transmission electron microscopy. When epithelial sheets were placed on intact basal laminae and incubated in the presence of the tyrosine kinase inhibitor genistein (200 microM), hemidesmosome formation was impaired. The formation of electron-dense regions was delayed, and no association of intermediate filaments was detected. Results were confirmed by biochemical studies. When the epithelium and underlying proteins were extracted and immunoprecipitated with beta 4 or BPAG-1, tyrosine phosphorylation decreased in the presence of genistein. In addition, the phosphorylation of beta 4 decreased when epithelial sheets were incubated on substrata from which the basal lamina had been removed or altered. Thus, a reduction in phosphorylation of tyrosine residues impairs the formation of mature hemidesmosomes, and substrata that fail to support hemidesmosome formation also demonstrate decreased phosphorylation of tyrosine residues.

Alkalies↗
PubMed2000

Comparative genomic hybridization reveals extensive variation among different MCF-7 cell stocks.

Comparative genomic hybridization (CGH) allows the detection of DNA sequence copy number changes on a genome-wide scale in a single hybridization reaction. The ability of CGH to be applied to formalin-fixed, paraffin-embedded tumor samples has lead to its widespread application in the cytogenetic analysis of archival material. When setting up CGH in the laboratory, rigorous control experiments must be carried out to ensure that the losses and gains are scored correctly. Groups interested in breast cancer frequently use the MCF-7 cell line as a positive control in these experiments, comparing the results to previously described genetic alterations. Here we present the results of CGH carried out with three stocks of MCF-7 cells. The cells differ widely in their proliferative response to 17-beta estradiol and show extensive variation in copy number changes affecting specific chromosomal regions. We suggest that care must be taken, therefore, when choosing a cell line as a positive control for CGH experiments.

Breast Neoplasms↗
PubMed2000

Prevention of yeast spoilage in feed and food by the yeast mycocin HMK.

The yeast Williopsis mrakii produces a mycocin or yeast killer toxin designated HMK; this toxin exhibits high thermal stability, high pH stability, and a broad spectrum of activity against other yeasts. We describe construction of a synthetic gene for mycocin HMK and heterologous expression of this toxin in Aspergillus niger. Mycocin HMK was fused to a glucoamylase protein carrier, which resulted in secretion of biologically active mycocin into the culture media. A partial purification protocol was developed, and a comparison with native W. mrakii mycocin showed that the heterologously expressed mycocin had similar physiological properties and an almost identical spectrum of biological activity against a number of yeasts isolated from silage and yoghurt. Two food and feed production systems prone to yeast spoilage were used as models to assess the ability of mycocin HMK to act as a biocontrol agent. The onset of aerobic spoilage in mature maize silage was delayed by application of A. niger mycocin HMK on opening because the toxin inhibited growth of the indigenous spoilage yeasts. This helped maintain both higher lactic acid levels and a lower pH. In yoghurt spiked with dairy spoilage yeasts, A. niger mycocin HMK was active at all of the storage temperatures tested at which yeast growth occurred, and there was no resurgence of resistant yeasts. The higher the yeast growth rate, the more effective the killing action of the mycocin. Thus, mycocin HMK has potential applications in controlling both silage spoilage and yoghurt spoilage caused by yeasts.

Aerobiosis↗
PubMed2000

Do eicosanoids cause colonic dysfunction in experimental E coli O157:H7 (EHEC) infection?

BACKGROUND: The pathophysiology of enterohaemorrhagic Escherichia coli (EHEC) infection remains unclear. Eicosanoids have been implicated as pathophysiological mediators in other colitides. AIMS: To determine if prostaglandin E(2) (PGE(2)) and leukotriene B(4) (LTB(4)) contribute to mucosal inflammation and dysfunction in EHEC colitis. METHODS: Ten day old rabbits were infected with EHEC. For five days after infection, mucosal synthesis of PGE(2) and LTB(4) was measured in distal colonic tissue from control and infected animals and (51)Cr-EDTA permeability was assessed in vivo. Myeloperoxidase activity was measured and histological inflammation and damage were assessed at five days in control and infected animals and after treatment of infected animals with the LTB(4) synthesis inhibitor MK-886. In separate experiments, ion transport was measured in Ussing chambers, before and after in vitro addition of the cyclooxygenase inhibitor indomethacin. RESULTS: LTB(4) synthesis was increased from day 2 after infection onwards and PGE(2) synthesis was increased on day 3. Mucosal permeability did not increase until day 5 after infection. MK-886 inhibited colonic LTB(4) production but did not reduce diarrhoea, inflammation, or mucosal damage. Electrolyte transport was not significantly altered on day 3 after infection. However, both Cl secretion and reduced Na absorption found on day 5 were partially reversed by indomethacin. CONCLUSIONS: Tissue synthesis of PGE(2) and LTB(4) did not correlate temporally with EHEC induced inflammation or changes in mucosal permeability and ion transport. Cyclooxygenase inhibition partially reversed ion transport abnormalities but lipoxygenase inhibition did not affect mucosal inflammation or histological damage. We conclude that the contribution of eicosanoids to mucosal injury and dysfunction is more complex than previously suggested.

Animals↗
PubMed2000

Prediction and assessment of the effects of mixtures of four xenoestrogens.

The assessment of mixture effects of estrogenic agents is regarded as an issue of high priority by many governmental agencies and expert decision-making bodies all over the world. However, the few mixture studies published so far have suffered from conceptual and experimental problems and are considered to be inconclusive. Here, we report the results of assessments of two-, three- and four-component mixtures of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol, all compounds with well-documented estrogenic activity. Extensive concentration-response analyses with the single agents were carried out using a recombinant yeast screen (yeast estrogen screen, YES). Based on the activity of the single agents in the YES assay we calculated predictions of entire concentration-response curves for mixtures of our chosen test agents assuming additive combination effects. For this purpose we employed the models of concentration addition and independent action, both well-established models for the calculation of mixture effects. Experimental concentration-response analyses revealed good agreement between predicted and observed mixture effects in all cases. Our results show that the combined effect of o,p'-DDT, genistein, 4-nonylphenol, and 4-n-octylphenol in the YES assay does not deviate from expected additivity. We consider both reference models as useful tools for the assessment of combination effects of multiple mixtures of xenoestrogens.

DDT↗
PubMed2000

Negative correlates of computer game play in adolescents.

There is some concern that playing computer games may be associated with social isolation, lowered self-esteem, and aggression among adolescents. Measures of these variables were included in a questionnaire completed by 204 year eight students at a North London comprehensive school. Principal components analysis of a scale to assess needs fulfilled by game play provided some support for the notion of 'electronic friendship' among boys, but there was no evidence that game play leads to social isolation. Play was not linked to self-esteem in girls, but a negative relationship was obtained between self-esteem and frequency of play in boys. However, self-esteem was not associated with total exposure to game play. Aggression scores were not related to the number of games with aggressive content named among three favourite games, but they were positively correlated with total exposure to game play. A multiple regression analysis revealed that sex and total game play exposure each accounted for a significant but small amount of the variance in aggression scores. The positive correlation between playing computer games and aggression provides some justification for further investigation of the causal hypothesis, and possible methodologies are discussed.

Adolescent↗
PubMed2000

Two alternative notions of health.

Contemporary health care as well as medical ethics deal with the problem of priorities while the invariable priority of any treatment across all cultures remains health. Health as such is, however, rather a symbolic word which refers to two different paradigms coined in two ancient traditions which contributed profoundly to European culture as we know it: the two traditions referred hereto are the Hebraic and the Greek. The Greek tradition considered health as a good life, which is the utmost goal of human endeavor. To achieve this it was necessary to contemplate truth and to become acquainted with certainty (episteme) beyond any doubt. On the contrary a totally different approach can be found in the Hebraic tradition which referred particularly to faith grasped as the hope (tikva) being based on a reliance upon the future ahead. Modern medicine hesitates between these two notions of health.

Culture↗
PubMed1999

Visualization and functional assessment of proximal and middle left anterior descending coronary stenoses in humans with magnetic resonance imaging.

BACKGROUND: Coronary artery bypass grafting improves survival in patients with >70% luminal diameter narrowing of the 3 major epicardial coronary arteries, particularly if there is involvement of the proximal portion of the left anterior descending (LAD) coronary artery. Measurement of coronary flow reserve can be used to identify functionally important luminal narrowing of the LAD artery. Although magnetic resonance imaging (MRI) has been used to visualize coronary arteries and to measure flow reserve noninvasively, the utility of MRI for detecting significant LAD stenoses is unknown. METHODS AND RESULTS: Thirty subjects (23 men, 7 women, age 36 to 77 years) underwent MRI visualization of the left main and LAD coronary arteries as well as measurement of flow in the proximal, middle, or distal LAD both at rest and after intravenous adenosine (140 microgram/kg per minute). Immediately thereafter, contrast coronary angiography and when feasible, intracoronary Doppler assessments of coronary flow reserve, were performed. There was a statistically significant correlation between MRI assessments of coronary flow reserve and (a) assessments of coronary arterial stenosis severity by quantitative coronary angiography and (b) invasive measurements of coronary flow reserve (P<0.0001 for both). In comparison to computer-assisted quantitative coronary angiography, the sensitivity and specificity of MRI for identifying a stenosis >70% in the distal left main or proximal/middle LAD arteries was 100% and 83%, respectively. CONCLUSIONS: Noninvasive MRI measures of coronary flow reserve correlated well with similar measures obtained with the use of intracoronary Doppler flow wires and predicted significant coronary stenoses (>70%) with a high degree of sensitivity and specificity. MRI-based measurement of coronary flow reserve may prove useful for identification of patients likely to obtain a survival benefit from coronary artery bypass grafting.

Adult↗
PubMed1999

Spatial and temporal trends and effects of contaminants in the Canadian Arctic marine ecosystem: a review.

Recent studies have added substantially to our knowledge of spatial and temporal trends of persistent organic pollutants and heavy metals in the Canadian Arctic marine ecosystem. This paper reviews the current state of knowledge of contaminants in marine biota in the Canadian Arctic and where possible, discusses biological effects. The geographic coverage of information on contaminants such as persistent organochlorines (OCs) (PCBs, DDT- and chlordane-related compounds, hexachlorocyclohexanes, toxaphene) and heavy metals (mercury, selenium, cadmium, lead) in tissues of marine mammal and sea birds is relatively complete. All major beluga, ringed seal and polar bear stocks along with several major sea bird colonies have been sampled and analysed for OC and heavy metal contaminants. Studies on contaminants in walrus are limited to Foxe Basin and northern Québec stocks, while migratory harp seals have only been studied recently at one location. Contaminant measurements in bearded seal, harbour seal, bowhead whale and killer whale tissues from the Canadian Arctic are very limited or non-existent. Many of the temporal trend data for contaminants in Canadian Arctic biota are confounded by changes in analytical methodology, as well as by variability due to age/size, or to dietary and population shifts. Despite this, studies of OCs in ringed seal blubber at Holman Island and in sea birds at Prince Leopold Island in Lancaster Sound show declining concentrations of PCBs and DDT-related compounds from the 1970s to 1980s then a levelling off during the 1980s and early 1990s. For other OCs, such as chlordane, HCH and toxaphene, limited data for the 1980s to early 1990s suggests few significant declines in concentrations in marine mammals or sea birds. Temporal trend studies of heavy metals in ringed seals and beluga found higher mean concentrations of mercury in more recent (1993/1994) samples than in earlier collections (1981-1984 in eastern Arctic, 1972-1973 in western Arctic) for both species. Rates of accumulation of mercury are also higher in present day animals than 10-20 years ago. Cadmium concentrations in the same animals (eastern Arctic only) showed no change over a 10-year period. No temporal trend data are available for metals in sea birds or polar bears. There have been major advances in knowledge of specific biomarkers in Canadian Arctic biota over the past few years. The species with the most significant risk of exposure to PCBs and OC pesticides may be the polar bear which, based on comparison with EROD activity in other marine mammals (beluga, ringed seal), appears to have elevated CYP1A-mediated activity. The MFO enzyme data for polar bear, beluga and seals suggest that even the relatively low levels of contaminants present in Arctic animals may not be without biological effects, especially during years of poor feeding.

Animals↗
PubMed1999

4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL)--An enzyme of phenylpropanoid chain cleavage from Pseudomonas.

The enzyme 4-hydroxycinnamoyl-CoA hydratase/lyase (HCHL), which catalyzes a hydration and two-carbon cleavage step in the degradation of 4-hydroxycinnamic acids, has been purified and characterized from Pseudomonas fluorescens strain AN103. The enzyme is a homodimer and is active with three closely related substrates, 4-coumaroyl-CoA, caffeoyl-CoA, and feruloyl-CoA (Km values: 5.2, 1.6, and 2.4 microM, respectively), but not with cinnamoyl-CoA or with sinapinoyl-CoA. The abundance of the enzyme reflects a low catalytic center activity (2.3 molecules s-1 at 30 degrees C; 4-coumaroyl-CoA as substrate).

Acyl Coenzyme A↗
PubMed1999

A role for the hepatobiliary system in IgE-mediated intestinal inflammation in the rat.

BACKGROUND: For many years the central focus of research into gastrointestinal hypersensitivity reactions has been the mast cell population of the intestinal lamina propria. Since bile is known to deliver immunological mediators to the gastrointestinal tract, the possibility arises that extra-intestinal populations of mast cells may also contribute to IgE-mediated intestinal damage. OBJECTIVES: To characterize hepatic mast cells in the rat and to investigate the role of the hepatobiliary system in a model of IgE-mediated reactivity to dietary antigen. METHODS: Wistar rats were passively sensitized with monoclonal antidinitrophenyl (DNP) IgE antibodies, and were later challenged orogastrically with DNP-HSA. Additional animals were sensitized, then bile duct-cannulated prior to antigen challenge. At various time points, liver and intestinal samples were collected for histological examination, and bile was collected and assayed for histamine and TNFalpha. RESULTS: Hepatic mast cells display a mucosal mast cell-like phenotype, and are closely associated with the vessels of the portal triads. Orogastric antigen challenge led to a rapid and significant decline (P<0.0001) in detectable mast cells as a result of anaphylactic degranulation. The median number of granulated mast cells associated with each portal triad in liver sections declined from six per portal triad to one per portal triad post-antigen challenge. After 15 min, biliary histamine concentrations rose above background levels (P<0.01). TNFalpha was also detectable in the majority (4/6) of bile samples within 15 min of challenge. Histological examination of the gastrointestinal mucosa revealed disruption to the villous epithelium ranging from oedematous changes to gross destruction. Such damage was not seen in animals in which bile had been externally drained. CONCLUSION: The data indicate that biliary products are major contributors to the gastrointestinal damage arising from IgE-mediated hypersensitivity reactions in the rat, and such hypersensitivity reactions may involve a population of mast cells which reside in the liver.

Animals↗
PubMed1999

Using multiple response true-false multiple choice questions.

The Royal Australasian College of Surgeons (RACS) Board of Examiners, having agreed to replace Type K with Type X multiple choice questions (MCQ), undertook a study to determine the most appropriate method of scoring the latter. Retrospective scoring of candidates' responses in a previous examination, using six reported scoring techniques for Type X items, showed that the Middlesex technique yielded a mean score closest to the mean score of the Type K items which were being replaced. However, as candidates' responses are affected by instructions, and the latter could not be controlled, a prospective study was carried out on three examinations, in each of which the nature of Part C varied among the three papers, while Parts A and B were similar. Paper 1 contained Type K items in Part C, Paper 2 contained Type X items with instructions appropriate for the Middlesex technique, and Paper 3 contained Type X items scored +1 if correct, -1 if incorrect and 0 if omitted. Items sampling the same content areas were randomly distributed among the three papers. The results indicated that the two Type X systems yielded significantly lower mean scores than Type K, probably due to combinations characteristic of the latter, enabling candidates with partial knowledge to obtain a maximum score. The difference in mean scores between the two Type X systems was not significant. A significantly higher mean percentage score in reused Type X (Middlesex system) items over that of the same items when they were previously used as Type K items was attributed to the combined effects of credit for partial knowledge, practice on reused items and differences in mean candidate ability across years. Pearson product-moment correlation coefficients between pairs of Part C mean percentage scores at each examination were positive and significant. The Board decided to replace Type K with Type X items, and the +1, 0, -1 system of scoring was adopted as it was considered more efficient than the Middlesex system, which did not give credit for identifying false statements as false.

Australia↗