Heterodimeric O-methyltransferases involved in the biosynthesis of noscapine in opium poppy.

Noscapine biosynthesis in opium poppy involves three characterized O-methyltransferases (OMTs) and a fourth responsible for the 4'-methoxyl on the phthalide isoquinoline scaffold. The first three enzymes are homodimers, whereas the latter is a heterodimer encoded by two linked genes (OMT2 and OMT3). Neither OMT2 nor OMT3 form stable homodimers, but yield a substrate-specific heterodimer when their genes are co-expressed in Escherichia coli. The only substrate, 4'-O-desmethyl-3-O-acetylpapaveroxine, is a seco-berbine pathway intermediate that undergoes ester hydrolysis subsequent to 4'-O-methylation leading to the formation of narcotine hemiacetal. In the absence of 4'-O-methylation, a parallel pathway yields narcotoline hemiacetal. Dehydrogenation produces noscapine and narcotoline from the corresponding hemiacetals. Phthalide isoquinoline intermediates with a 4'-hydroxyl (i.e. narcotoline and narcotoline hemiacetal), or the corresponding 1-hydroxyl on protoberberine intermediates, were not accepted. Norcoclaurine 6OMT, which shares 81% amino acid sequence identity with OMT3, also formed a functionally similar heterodimer with OMT2. Suppression of OMT2 transcript levels in opium poppy increased narcotoline accumulation, whereas reduced OMT3 transcript abundance caused no detectable change in the alkaloid phenotype. Opium poppy chemotype Marianne accumulates high levels of narcotoline and showed no detectable OMT2:OMT3 activity. Compared with the active subunit from the Bea's Choice chemotype, Marianne OMT2 exhibited a single S122Y mutation in the dimerization domain that precluded heterodimer formation based on homology models. Both subunits contributed to the formation of the substrate-binding domain, although site-directed mutagenesis revealed OMT2 as the active subunit. The occurrence of physiologically relevant OMT heterodimers increases the catalytic diversity of enzymes derived from a smaller number of gene products.

Mechanical environment, donor age, and presence of endothelium interact to modulate porcine artery viability ex vivo.

Though ex vivo culture of arteries is a widely used model of native arteries and is closely aligned with efforts to generate tissue-engineered arteries, the effects of culture conditions on artery viability are poorly characterized. To investigate factors regulating long-term viability of cultured arteries, carotid arteries from neonatal and adolescent pigs were perfused for up to 27 days with steady laminar flow ranging from approximately 2% to approximately 200% of physiological flow rates. Arteries from neonatal animals (2 weeks old, approximately 5 kg) were susceptible to spontaneous progressive endothelial denudation followed by deterioration of the vessel wall that spread from luminal to abluminal regions. Subphysiological levels of flow and pressure abrogated this deterioration. Arteries harvested from adolescent (6 months old, approximately 100 kg) animals maintained viability and retained structure for at least 9 days as assessed by normal histology, presence of intact endothelium, normal mitochondrial activity, and low levels of cell death and proliferation, unless the vessels were subjected to superphysiological levels of flow or the endothelium was intentionally denuded. Adolescent arteries perfused at subphysiological, but not physiological, flow rates maintained viability and normal structure for at least 27 days. These data indicate that under the appropriate conditions, arteries may be cultured long term but careful attention to the viability is merited.

The role of friction in the measurement of slipperiness, Part 1: friction mechanisms and definition of test conditions.

Friction has been widely used as a measure of slipperiness. However, controversies around friction measurements remain. The purposes of this paper are to summarize understanding about friction measurement related to slipperiness assessment of shoe and floor interface and to define test conditions based on biomechanical observations. In addition, friction mechanisms at shoe and floor interface on dry, liquid and solid contaminated, and on icy surfaces are discussed. It is concluded that static friction measurement, by the traditional use of a drag-type device, is only suitable for dry and clean surfaces, and dynamic and transition friction methods are needed to properly estimate the potential risk on contaminated surfaces. Furthermore, at least some of the conditions at the shoe/floor interface during actual slip accidents should be replicated as test conditions for friction measurements, such as sliding speed, contact pressure and normal force build-up rate.

The role of friction in the measurement of slipperiness, Part 2: survey of friction measurement devices.

This paper seeks to address questions related to friction measurement such as how friction is related to human-centred assessment and actual slipping, and how repeatable friction measurements are. Commonly used devices for slipperiness measurement are surveyed and their characteristics compared with suggested test conditions from biomechanical observations summarized in Part 1. The issues of device validity, repeatability, reproducibility and usability are examined from the published literature. Friction assessment using the mechanical measurement devices described appears generally valid and reliable. However, the validity of most devices could be improved by bringing them within the range of human slipping conditions observed in biomechanical studies. Future studies should clearly describe the performance limitations of any device and its results and should consider whether the device conditions reflect these actual human slipping conditions. There is also a need for validation studies of more devices by walking experiments.

The effect of load carrying and floor contaminants on slip and fall parameters.

A study was conducted to provide set-up values of an important biomechanical parameter, heel velocity, for slip resistance testers. Four different floor surfaces (with or without oil contaminant) were prepared for ten subjects with each walking at a fixed velocity while carrying five different loads. Stride lengths were also measured to find whether a significant difference existed between a carrying method typically used in industry (two-handed carrying of a load in a tote box held directly in front of the body) and previously used load carrying techniques (backpack and front-back pack systems). A programmable slip resistance tester was used to measure dynamic coefficient of friction with conventional set-up values for heel velocity. The results showed that the conventional set-up value for heel velocity is valid for dry floors but is too low on oily floors because faster transfer of body weight was seen owing to decreased stride length. On oily floors, heel velocities of 60 to 140 cm/s would be recommended. An abnormal gait pattern, short stride length, was seen on oily floors or with heavy load carriage because subjects adjusted their stride length for a better stance.