Molecular basis of copper transport: cellular and physiological functions of Menkes and Wilson disease proteins (ATP7A and ATPB)

Eukaryotic cells prevent copper-induced, free radical damage to cell components by employing copper-binding proteins and transporters that minimize the likelihood of free copper ions existing in the cell. In the cell, copper is actively transported from the cytoplasm during the biosynthesis of secreted coppercontaining proteins and, as a protective measure, when there is an excess of copper. In humans, this is accomplished by two related copper-transporting ATPases (ATP7A and ATP7B), which are the affected genes in two distinct human genetic disorders of copper transport, Menkes disease (copper deficiency) and Wilson disease (copper toxicosis). The study of these ATPases has revealed their molecular mechanisms of copper transport and their roles in physiological copper homeostasis. Both ATP7A and ATP7B are expressed in specific brain regions and neurological abnormalities are important clinical features in Menkes and Wilson disease.

The molecular basis of copper-transport diseases

Copper (Cu) is a potentially toxic yet essential element. Menkes disease, a copper deficiency disorder, and Wilson disease, a copper toxicosis condition, are two human genetic disorders, caused by mutations of two closely related Cu-transporting ATPases. Both molecules efflux copper from cells. Quite diverse clinical phenotypes are produced by different mutations of these two Cu-transporting proteins. The understanding of copper homeostasis has become increasingly important in clinical medicine as the metal could be involved in the pathogenesis of some important neurological disorders such as Alzheimer's disease, motor neurone diseases and prion diseases.

Modelling solute transport in structured soils: performance evaluation of the ADR and TRM models

The movement of chemicals through the soil to the groundwater or discharged to surface waters represents a degradation of these resources. In many cases, serious human and stock health implications are associated with this form of pollution. The chemicals of interest include nutrients, pesticides, salts, and industrial wastes. Recent studies have shown that current models and methods do not adequately describe the leaching of nutrients through soil, often underestimating the risk of groundwater contamination by surface-applied chemicals and overestimating the concentration of resident solutes. This inaccuracy results primarily from ignoring soil structure and nonequilibrium between soil constituents, water, and solutes. A multiple sample percolation system (MSPS), consisting of 25 individual collection wells, was constructed to study the effects of localized soil heterogeneities on the transport of nutrients (NO−3, Cl−, PO3−4) in the vadose zone of an agricultural soil predominantly dominated by clay. Very significant variations in drainage patterns across a small spatial scale were observed (one-way ANOVA, p < 0.001 indicating considerable heterogeneity in water flow patterns and nutrient leaching. Using data collected from the multiple sample percolation experiments, this paper compares the performance of two mathematical models for predicting solute transport, the advective-dispersion model with a reaction term (ADR), and a two-region preferential flow model (TRM) suitable for modelling nonequilibrium transport. These results have implications for modelling solute transport and predicting nutrient loading on a larger scale.

Fasting activates the gene expression of UCP3 independent of genes necessary for lipid transport and oxidation in skeletal muscle

Fasting triggers a complex array of adaptive metabolic and hormonal responses including an augmentation in the capacity for mitochondrial fatty acid (FA) oxidation in skeletal muscle. This study hypothesized that this adaptive response is mediated by increased mRNA of key genes central to the regulation of fat oxidation in human skeletal muscle. Fasting dramatically increased UCP3 gene expression, by 5-fold at 15 h and 10-fold at 40 h. However the expression of key genes responsible for the uptake, transport, oxidation, and re-esterification of FA remained unchanged following 15 and 40 h of fasting. Likewise there was no change in the mRNA abundance of transcription factors. This suggests a unique role for UCP3 in the regulation of FA homeostasis during fasting as adaptation to 40 h of fasting does not require alterations in the expression of other genes necessary for lipid metabolism.

Correction of the copper transport defect of Menkes patient fibroblasts by expression of two forms of the sheep Wilson ATPase

The Wilson disease (WD) protein (ATP7B) is a copper-transporting P-type ATPase that is responsible for the efflux of hepatic copper into the bile, a process that is essential for copper homeostasis in mammals. Compared with other mammals, sheep have a variant copper phenotype and do not efficiently excrete copper via the bile, often resulting in excessive copper accumulation in the liver. To investigate the function of sheep ATP7B and its potential role in the copper-accumulation phenotype, cDNAs encoding the two forms of ovine ATP7B were transfected into immortalised fibroblast cell lines derived from a Menkes disease patient and a normal control. Both forms of ATP7B were able to correct the copper-retention phenotype of the Menkes cell line, demonstrating each to be functional copper-transporting molecules and suggesting that the accumulation of copper in the sheep liver is not due to a defect in the copper transport function of either form of sATP7B.

Molecular and cellular aspects of copper transport in developing mammals

Copper is an essential trace element that requires tightly regulated homeostatic mechanisms to ensure adequate supplies without any toxic effects because of the ability of the metal ion to catalyze the formation of free radicals. The Cu-ATPases, ATP7A and ATP7B, play an important role in the physiological regulation of copper. Adequate supplies of copper are particularly important in developing animals, and in humans this is illustrated by mutations of ATP7A that cause the copper deficiency condition Menkes disease, which is fatal in early childhood. In contrast, mutations in ATP7B result in the genetic toxicosis, Wilson disease. We propose that the physiological regulation of copper is accomplished mainly by the intracellular copper-regulated trafficking of the Cu-ATPases. This process allows the overall copper status in the body to be maintained when levels of copper in the diet alter. A study of the defects in mouse models of Menkes and Wilson diseases has demonstrated that both ATPases play an important role in supplying copper to the developing fetus and neonate

Psychological and situational influences on commuter-transport-mode choice

The relative importance and relationship between psychological and situational factors in predicting commuter-transport-mode choice was tested by four hypotheses. First, the influence of individuals’ values on commuter behavior is mediated by their corresponding beliefs about the environmental threat of cars (mediation hypothesis). Second, the influence of these beliefs on behavior is moderated by individual consideration of future consequences and control beliefs (moderation hypothesis). Third, cost, time, and access factors contribute to individuals’ commuter choice (situational hypothesis). Fourth, situational and psychological factors jointly influence proenvironmental behavior (interaction hypothesis). A sample of 205 Australian university students completed a survey to measure these relationships. Regression analyses indicated support for the mediation, situational, and interaction hypotheses. It was concluded that to achieve a transport-mode shift to public transport, public policy strategies should focus on individuals’ transport-related environmental beliefs (personal control and environmental effect of cars) and situations (access to public transport at reduced cost).

Active transport to work in Australia: is it all downhill from here?

Physical inactivity is increasing in Australia and active forms of transportation may be one way to increase the working population’s daily physical activity. We used travel-to-work data from employed persons aged 15 years and over participating in the 1996 (n=7,636,319) and 2001(n=8,298,606) Australian censuses to determine prevalence and trends in walking and cycling to work by state and gender, and differences in prevalence by age. In 2001, 3.8% of Australians walked to work and <1% cycled. Over 64% travelled to work by car. There have been small declines in walking (men and women) and cycling (men) over the 5-years from 1996 to 2001. People were more likely to walk or cycle to work if they lived in the Northern Territory, if they were male or if they were aged 15 to 24 years. They were more likely to travel by car if they lived in the Australian Capital Territory, if they were male, or if they were aged 45-54 years. Few people walk or cycle to work in Australia. Efforts to encourage active transportation are urgently needed.

Nocodazole inhibits insulin-stimulated glusocs transport in 3T3-L1 adipocytes via a microtubule independant mechanism

Insulin stimulates glucose transport in adipocytes and muscle cells by triggering redistribution of the GLUT4 glucose transporter from an intracellular perinuclear location to the cell surface. Recent reports have shown that the microtubule-depolymerizing agent nocodazole inhibits insulin-stimulated glucose transport, implicating an important role for microtubules in this process. In the present study we show that 2 µM nocodazole completely depolymerized microtubules in 3T3-L1 adipocytes, as determined morphologically and biochemically, resulting in dispersal of the perinuclear GLUT4 compartment and the Golgi apparatus. However, 2 µM nocodazole did not significantly effect either the kinetics or magnitude of insulin-stimulated glucose transport. Consistent with previous studies, higher concentrations of nocodazole (10-33 µM) significantly inhibited basal and insulin-stimulated glucose uptake in adipocytes. This effect was not likely the result of microtubule depolymerization because in the presence of taxol, which blocked nocodazole-induced depolymerization of microtubules as well as the dispersal of the perinuclear GLUT4 compartment, the inhibitory effect of 10-33 µM nocodazole on insulin-stimulated glucose uptake prevailed. Despite the decrease in insulin-stimulated glucose transport with 33 µM nocodazole we did not observe inhibition of insulin-stimulated GLUT4 translocation to the cell surface under these conditions. Consistent with a direct effect of nocodazole on glucose transporter function we observed a rapid inhibitory effect of nocodazole on glucose transport activity when added to either 3T3-L1 adipocytes or to Chinese hamster ovary cells at 4 °C. These studies reveal a new and unexpected effect of nocodazole in mammalian cells which appears to occur independently of its microtubule-depolymerizing effects.

Public transport and pedestrian access, the impact of suburban planning on adolescence

The following paper discusses the impact of suburban life for adolescents in relation to public transport and pedestrian access. The research compares the morphology and level of connectivity of two planning models, cul-de-sac and regular grid, with respect to two suburbs which share a similar socio-economic profile, Rowville and Caulfield. These are subsequently evaluated in terms of ABS statistics, in particular year 12 or equivalent levels of education, and transport statistics. The results of the investigation are discussed in reference to the four key tasks of adolescence (Carr-Greg & Shale 2002)', in order to establish the degree to which morphology and access levels of suburbs impact on adolescent development.

Transport of cryptotanshinone, a major active triterpenoid in Salvia miltiorrhiza Bunge widely used in the treatment of stroke and Alzheimers disease, across the blood-brain

Cryptotanshinone (CTS), a major constituent from the roots of Salvia miltiorrhiza (Danshen), is widely used in the treatment of coronary heart disease, stroke and less commonly Alzheimer's disease. Our recent study indicates that CTS is a substrate for Pglycoprotein (PgP/MDR1/ABCB1). This study has investigated the nature of the brain distribution of CTS across the brain-blood barrier (BBB) using several in vitro and in vivo rodent models. A polarized transport of CTS was found in rat primary microvascular endothelial cell (RBMVEC) monolayers, with facilitated efflux from the abluminal side to luminal side. Addition of a PgP (e.g. verapamil and quinidine) or multi-drug resistance protein 1/2 (MRP1/2) inhibitor (e.g. probenecid and MK-571) in both luminal and abluminal sides attenuated the polarized transport. In a bilateral in situ brain perfusion model, the uptake of CTS into the cerebrum increased from 0.52 ± 0.1% at 1 min to 11.13 ± 2.36 ml/100 g tissue at 30 min and was significantly greater than that of sucrose. Co-perfusion of a PgP/MDR1 (e.g. verapamil) or MRP1/2 inhibitor (e.g. probenecid) significantly increased the brain distribution of CTS by 35.1-163.6%. The brain levels of CTS were only about 21% of those in plasma, and were significantly increased when coadministered with verapamil or probenecid in rats. The brain levels of CTS in rats subjected to middle cerebral artery occlusion and rats treated with quinolinic acid (a neurotoxin) were about 2- to 2.5-fold higher than the control rats. Moreover, the brain levels in mdr1a(-/-) and mrp1(-/-) mice were 10.9- and 1.5-fold higher than those in the wild-type mice, respectively. Taken collectively, these findings indicate that PgP and Mrp1 limit the brain penetration of CTS in rodents, suggesting a possible role of PgP and MRP1 in limiting the brain penetration of CTS in patients and causing drug resistance to Danshen therapy and interactions with conventional drugs that are substrates of PgP and MRP1. Further studies are needed to explore the role of other drug transporters in restricting the brain penetration of CTS and the clinical relevance.