Mutation analysis of the CDKN2A promoter in Australian melanoma families

Approximately 50% of all melanoma families worldwide show linkage to 9p21-22, but only about half of these have been shown to contain germ line CDKN2A mutations. It has been hypothesized that a proportion of these families carry mutations in the noncoding regions of CDKN2A. Several Canadian families have been reported to carry a mutation in the 5' UTR, at position -34 relative to the start site, which gives rise to a novel AUG translation initiation codon that markedly decreases translation from the wild-type AUG (Liu et al., 1999). Haplotype sharing in these Canadian families suggested that this mutation is of British origin. We sequenced 1,327 base pairs (bp) of CDKN2A, making up 1,116 bp of the 5' UTR and promoter, all of exon 1, and 61 bp of intron 1, in at least one melanoma case from 110 Australian families with three or more affected members known not to carry mutations within the p16 coding region. In addition, 431 bp upstream of the start codon was sequenced in an additional 253 affected probands from two-case melanoma families for which the CDKN2A mutation status was unknown. Several known polymorphisms at positions -33, -191, -493, and -735 were detected, in addition to four novel variants at positions 120, -252, -347, and -981 relative to the start codon. One of the probands from a two-case family was found to have the previously reported Q50R mutation. No family member was found to carry the mutation at position -34 or any other disease-associated mutation. For further investigation of noncoding CDKN2A mutations that may affect transcription, allele-specific expression analysis was carried out in 31 of the families with at least three affected members who showed either complete or indeterminate 9p haplotype sharing without CDKN2A exonic mutations. Reverse transcription polymerase chain reaction and automated sequencing showed expression of both CDKN2A alleles in all family members tested. The lack of CDKN2A promoter mutations and the absence of transcriptional silencing in the germ line of this cohort of families suggest that mutations in the promoter and 5' UTR play a very limited role in melanoma predisposition. (C) 2001 Wiley-Liss, Inc.

A dynamic model with on-line identification for rotary sugar drying processes

A dynamic modelling methodology, which combines on-line variable estimation and parameter identification with physical laws to form an adaptive model for rotary sugar drying processes, is developed in this paper. In contrast to the conventional rate-based models using empirical transfer coefficients, the heat and mass transfer rates are estimated by using on-line measurements in the new model. Furthermore, a set of improved sectional solid transport equations with localized parameters is developed in this work to reidentified on-line using measurement data, the model is able to closely track the dynamic behaviour of rotary drying processes within a broad range of operational conditions. This adaptive model is validated against experimental data obtained from a pilot-scale rotary sugar dryer. The proposed modelling methodology can be easily incorporated into nonlinear model based control schemes to form a unified modelling and control framework.place the global correlation for the computation of solid retention time. Since a number of key model variables and parameters are identified on-line using measurement data, the model is able to closely track the dynamic behaviour of rotary drying processes within a broad range of operational conditions. This adaptive model is validated against experimental data obtained from a pilot-scale rotary sugar dryer. The proposed modelling methodology can be easily incorporated into nonlinear model based control schemes to form a unified modelling and control framework.

Quantitative and qualitative influences of tapasin on the class I peptide repertoire

Tapasin is critical for efficient loading and surface expression of most HLA class I molecules. The high level surface expression of HLA-B*2705 on tapasin-deficient 721.220 cells allowed the influence of this chaperone on peptide repertoire to be examined. Comparison of peptides bound to HLA-B*2705 expressed on tapasin-deficient and -proficient cells by mass spectrometry revealed an overall reduction in the recovery of B*2705-bound peptides isolated from tapasin-deficient cells despite similar yields of B27 heavy chain and beta (2)-microglobulin. This indicated that a proportion of suboptimal ligands were associated with B27, and they were lost during the purification process. Notwithstanding this failure to recover these suboptimal peptides, there was substantial overlap in the repertoire and biochemical properties of peptides recovered from B27 complexes derived from tapasin-positive and -negative cells. Although many peptides were preferentially or uniquely isolated from B*2705 in tapasin-positive cells, a number of species were preferentially recovered in the absence of tapasin, and some of these peptide ligands have been sequenced. In general, these ligands did not exhibit exceptional binding affinity, and we invoke an argument based on lumenal availability and affinity to explain their tapasin independence. The differential display of peptides in tapasin-negative and -positive cells was also apparent in the reactivity of peptide-sensitive alloreactive CTL raised against tapasin-positive and -negative targets, demonstrating the functional relevance of the biochemical observation of changes in peptide repertoire in the tapasin-deficient APC. Overall, the data reveal that tapasin quantitatively and qualitatively influences ligand selection by class I molecules.

Incubation temperature, energy expenditure and hatchling size in the green turtle (Chelonia mydas), a species with temperature-sensitive sex determination

Eggs from the Heron Island, Great Barrier Reef, nesting population of green turtles (Chelonia mydas) were incubated at all-male-determining (26 degreesC) and all-female-determining (30 degreesC) temperatures. Oxygen consumption and embryonic growth were monitored throughout incubation, and hatchling masses and body dimensions were measured from both temperatures. Eggs hatched after 79 and 53 days incubation at 26 degreesC and 30 degreesC respectively. Oxygen consumption at both temperatures increased to a peak several days before hatching, a pattern typical of turtle embryos, and the rate of oxygen was higher at 30 degreesC than 26 degreesC. The total amount of energy consumed during incubation, and hatchling dimensions, were similar at both temperatures, but hatchlings from 26 degreesC had larger mass, larger yolk-free mass and smaller residual yolks than hatchlings from 30 degreesC. Because of the difference in mass of hatchlings, hatchlings from 30 degreesC had a higher production cost.

The effect of obstacle position on reach-to-grasp movements

Numerous everyday tasks require the nervous system to program a prehensile movement towards a target object positioned in a cluttered environment. Adult humans are extremely proficient in avoiding contact with any non-target objects (obstacles) whilst carrying out such movements. A number of recent studies have highlighted the importance of considering the control of reach-to-grasp (prehension) movements in the presence of such obstacles. The current study was constructed with the aim of beginning the task of studying the relative impact on prehension as the position of obstacles is varied within the workspace. The experimental design ensured that the obstacles were positioned within the workspace in locations where they did not interfere physically with the path taken by the hand when no obstacle was present. In all positions, the presence of an obstacle caused the hand to slow down and the maximum grip aperture to decrease. Nonetheless, the effect of the obstacle varied according to its position within the workspace. In the situation where an obstacle was located a small distance to the right of a target object, the obstacle showed a large effect on maximum grip aperture but a relatively small effect on movement time. In contrast, an object positioned in front and to the right of a target object had a large effect on movement speed but a relatively small effect on maximum grip aperture. It was found that the presence of two obstacles caused the system to decrease further the movement speed and maximum grip aperture. The position of the two obstacles dictated the extent to which their presence affected the movement parameters. These results show that the antic ipated likelihood of a collision with potential obstacles affects the planning of movement duration and maximum grip aperture in prehension.

Development of a real-time RT-PCR assay for plasma membrane calcium ATPase isoform 1 (PMCA1) mRNA levels in a human breast epithelial cell line.

The plasma membrane Ca2+ pump is a key regulator of cytosolic free Ca2+. Recent studies have demonstrated the dynamic expression of the plasma membrane Ca2+ pump in a variety of cell types. Furthermore, alterations in plasma membrane calcium pump activity have now been implicated in human disease. In this study, the development of a technique to quantitatively assess mRNA expression of the human plasma membrane Ca2+ ATPase (PMCA1) isoform of the plasma membrane Ca2+ pump, using a real-time reverse transcriptase-polymerase chain reaction (real-time RT-PCR) assay in a human breast epithelial cell line (MCF-7) is described. The sequences of the PMCA1 primers and probe for real-time RT-PCR are presented. The results also indicate that PMCA1 mRNA can be normalized to both 18S ribosomal RNA (18S rRNA) and human glyceraldehyde-3-phosphate dehydrogenase (hGAPDH) in MCF-7 cells. Real-time RT-PCR will be most useful in assessing PMCA1 mRNA expression in cases where only low amounts of RNA are available and/or when numerous samples must be assessed simultaneously. (C) 2001 Elsevier Science Inc. All rights reserved.

From idola conceptiones to discourse analysis: A response to Bart Crum

Crum's notion of idola as a conceptual fallacy is interesting, somewhat helpful, yet potentially limiting in a critique of research in PE if one is to accept a postmodern or poststructuralist position. In line with a poststructuralist position, a strength in Crum's application of idola is the recognition that research in PE is constituted by the researcher and their social world which, in turn, constitutes the researcher. Limitations to Crum's idola thesis arise when the notion is used to suggest that as a result of the researcher's lack of conceptual clarity, the quest for knowledge or truth about PE pedagogy is undermined as this assumes that meanings can be unequivocal and precede a linear research process. In contrast, this response argues that a priority in research should be to examine how, and under what conditions, particular discourses come to shape PE practices in schools and universities. In a postmodern world, conceptual clarity should not be the goal but rather a coming to understand how PE knowledge and practice is being constructed across sites and contexts.

SOX9 enhances aggrecan gene promoter/enhancer activity and is up-regulated by retinoic acid in a cartilage-derived cell line, TC6

SOX9 is a transcription factor that plays a key role in chondrogenesis, Aggrecan is one of the major structural components in cartilage; however, the molecular mechanism of aggrecan gene regulation has not yet been fully elucidated, TC6 is a clonal chondrocytic cell line derived from articular cartilage, The purpose of this study was to examine whether SOX9 modulates aggrecan gene expression and to further identify molecules that regulate Sox9 expression in TC6 cells. SOX9 overexpression in TC6 cells enhanced by similar to 3-fold the transcriptional activity of the AgCAT-8 construct containing S-kilobase (kb) promoter/first exon/first intron fragments of the aggrecan gene. SOX9 enhancement of aggrecan promoter activity was lost when we deleted a 4.5-kb fragment from the 3'-end of the 8-kb fragment corresponding to the region including the first intron, In TC6 cells, SOX9 enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence >10-fold. SOX9 enhancement of aggrecan gene promoter activity and SOX9 transactivation through the Sry/Sox consensus sequence were not observed in osteoblastic osteosarcoma cells (ROS17/2.8), indicating the dependence on the cellular background. Northern blot analysis indicated that TC6 cells constitutively express Sox9 mRNA at relatively low levels. To examine regulation of Sox9 gene expression, we investigated the effects of calciotropic hormones and cytokines, Among these, retinoic acid (RA) specifically enhanced Sox9 mRNA expression in TC6 cells. The basal levels of Sox9 expression and its enhancement by RA were observed similarly at both permissive (33 degrees C) and nonpermissive (39 degrees C) temperatures. Furthermore, RA treatment enhanced the transcriptional activity of a reporter construct containing the Sry/Sox consensus sequence in TC6 cells. Moreover, RA treatment also enhanced the transcriptional activity of another reporter construct containing the enhancer region of the type II procollagen gene in TC6 cells. These observations indicate that SOX9 enhances aggrecan promoter activity and that its expression is up-regulated by RA in TC6 cells.

The effects of time delays in adaptive phase measurements

It is not possible to make measurements of the phase of an optical mode using linear optics without introducing an extra phase uncertainty. This extra phase variance is quite large for heterodyne measurements, however it is possible to reduce it to the theoretical limit of log (n) over bar (4 (n) over bar (2)) using adaptive measurements. These measurements are quite sensitive to experimental inaccuracies, especially time delays and inefficient detectors. Here it is shown that the minimum introduced phase variance when there is a time delay of tau is tau/(8 (n) over bar). This result is verified numerically, showing that the phase variance introduced approaches this limit for most of the adaptive schemes using the best final phase estimate. The main exception is the adaptive mark II scheme with simplified feedback, which is extremely sensitive to time delays. The extra phase variance due to time delays is considered for the mark I case with simplified feedback, verifying the tau /2 result obtained by Wiseman and Killip both by a more rigorous analytic technique and numerically.

Phase sensitive pumping and coherence of superposition states

The continuous parametric pumping of a superconducting lossy QED cavity supporting a field prepared initially as a superposition of coherent states is discussed. In contrast to classical pumping, we verify that the phase sensitivity of the parametric pumping makes the asymptotic behaviour of the cavity field state strongly dependent on the phase theta of the coherent state \ alpha > = \ alpha \e(i theta)>. Here we consider theta = pi /4, -pi /4 and we analyse the evolution of the purity of the superposition states with the help of the linear entropy and fidelity functions. We also analyse the decoherence process quantitatively through the Wigner function, for both states, verifying that the decay is slightly modified when compared to the free decoherence case: for theta = -pi /4 the process is accelerated while for theta = pi /4 it is delayed.

The management of rice grown on raised beds with continuous furrow irrigation

A field experiment compared two rice (Oryza sativa L.) cropping systems: paddy or raised beds with continuous furrow irrigation; and trialled four cultivars: Starbonnet, Lemont, Amaroo and Ceysvoni, and one test line YRL39; that may vary in adaptation to growth on raised beds. The grain yield of rice ranged from 740 to 1250 g/m(2) and was slightly greater in paddy than on raised beds. Although there were early growth responses to fertilizer nitrogen on raised beds, the crop nitrogen content at maturity mostly exceeded 20 g/m(2) in both systems, so nitrogen was unlikely to have limited yield. Ceysvoni yielded best in both systems, a result of good post-anthesis growth and larger grain size, although its whole-grain mill-out percentage was poor relative to the other cultivars. Starbonnet and Lemont yielded poorly on raised beds, associated with too few tillers and too much leaf area. When grown on raised beds all cultivars experienced a delay in anthesis resulting in more tillers, leaf area and dry weight at anthesis, and probably a greater yield potential. The growth of rice after anthesis, however, was similar on raised beds and in paddy, so reductions in harvest index and grain size on raised beds were recorded. The data indicated that water supply was not a major limitation to rice growth on raised beds, but slower crop development was an issue that would affect the use of raised beds in a cropping system, especially in rice-growing areas where temperatures are too cool for optimal crop development. (C) 2001 Elsevier Science B.V. All rights reserved.

Size and power properties of the AH test of evolutionary change

We investigate the size and power properties of the AH test of evolutionary change. This involves examining whether the size results are sensitive to both the number of individual frequencies estimated and the spectral shape adopted under the null hypothesis. The power tests examine whether the test has good power to detect shifts in both spectral position (variance) and spectral shape (autocovariance structure).