Effect of the toxic milk mutation (tx) on the function and intracellular localization of Wnd, the murine homologue of the Wilson copperATPase

Wilson disease is an autosomal recessive copper transport disorder resulting from defective biliary excretion of copper and subsequent hepatic copper accumulation and liver failure if not treated. The disease is caused by mutations in the ATP7B (WND) gene, which is expressed predominantly in the liver and encodes a copper-transporting P-type ATPase that is structurally and functionally similar to the Menkes protein (MNK), which is defective in the X-linked copper transport disorder Menkes disease. The toxic milk (tx) mouse has a clinical phenotype similar to Wilson disease patients and, recently, the tx mutation within the murine WND homologue (Wnd) of this mouse was identified, establishing it as an animal model for Wilson disease. In this study, cDNA constructs encoding the wild-type (Wnd-wt) and mutant (Wnd-tx) Wilson proteins (Wnd) were generated and expressed in Chinese hamster ovary (CHO) cells. The tx mutation disrupted the copper-induced relocalization of Wnd in CHO cells and abrogated Wnd-mediated copper resistance of transfected CHO cells. In addition, co-localization experiments demonstrated that while Wnd and MNK are located in the trans-Golgi network in basal copper conditions, with elevated copper, these proteins are sorted to different destinations within the same cell. Ultrastructural studies showed that with elevated copper levels, Wnd accumulated in large multi-vesicular structures resembling late endosomes that may represent a novel compartment for copper transport. The data presented provide further support for a relationship between copper transport activity and the copper-induced relocalization response of mammalian copper ATPases, and an explanation at a molecular level for the observed phenotype of tx mice

Consumers' health perceptions of three types of milk: a survey in Australia

A survey was conducted among 345 randomly selected shoppers in Melbourne, Australia, to identify their perceptions of the healthiness of whole milk, reduced fat milk and soy milk and to investigate demographic influences on health perceptions and types of milk consumption. The survey revealed major differences in shoppers' perceptions of the three types of milk along the themes of bone health, weight control, disease prevention, and, allergy and disease causation. Generally whole milk was perceived more negatively than soy or reduced fat milk. There were few demographic differences in the shoppers' perceptions of the three products. Reported consumption of reduced fat milk was more frequent among women and elderly people, and whole milk consumption was positively related to parenthood. The findings suggest that previous marketing and education campaigns have been effective but the widespread uncertainty and erroneous beliefs about milk products suggest that many consumers have not assimilated new findings about milk products. This represents a challenge for industry and public health practitioners.

Consumers` sensory and nutritional perceptions of three types of milk

Objective: To identify consumer perceptions of whole milk, reduced-fat milk and soy milk, and to investigate demographic influences on perceptions and types of milk consumption.

Design and setting: Questionnaires covering nutritional and sensory perceptions of three types of milk.

Subjects: Three hundred and sixty-one randomly selected shoppers in Melbourne, Australia.

Results: Generally, respondents held positive perceptions about milk. Milk was considered as having good sensory properties, providing a good source of nutrients, and being a convenient and safe product. However, despite these findings, misperceptions and unawareness about the nutrient content of milk were prevalent. Negative perceptions were most common for whole milk and were mostly related to its perceived high fat, cholesterol and energy contents. Soy milk received lower ratings on sensory quality and convenience than dairy milk. There were few sociodemographic differences in consumers' perceptions. Although reduced-fat milk consumption was more frequent among elderly people and type of milk consumption was related to parenthood, no other significant effects of demographic variables were found on the consumption of specific milk types.

Conclusion: Although positive perceptions were common, negative perceptions and misperceptions appear to be prevalent, presenting a challenge for nutrition education. Sociodemographic factors were not shown to be important predictors of perceptions and type of milk consumption.

Analysis of zinc transporter, hZnT4(Slc30A4), gene expression in a mammary gland disorder leading to reduced zinc secretion into milk

Zinc deficiency, causing impaired growth and development, may have a nutritional or genetic basis. We investigated two cases of inherited zinc deficiency found in breast-fed neonates, caused by low levels of zinc in the maternal milk. This condition is different from acrodermatitis enteropathica but has similarities to the "lethal milk" mouse, where low levels of zinc in the milk of lactating dams leads to zinc deficiency in pups. The mouse disorder has been attributed to a defect in the ZnT4 gene. Little is known about the expression of the human orthologue, hZnT4 (Slc30A4). Sequence analysis of cDNA, real-time PCR and Western blot analysis of hZnT4, carried out on control cells and cells from unrelated mothers of two infants with zinc deficiency, showed no differences. The hZnT4 gene was highly expressed in mouthwash buccal cells compared with lymphoblasts and fibroblasts. The hZnT4 protein did not co-localise with intracellular free zinc pools, suggesting that hZnT4 is not involved in transport of zinc into vesicles destined for secretion into milk. This observation, combined with phenotypic differences between the "lethal milk" mouse and the human disorder, suggests that the "lethal milk" mouse is not the corresponding model for the human zinc deficiency condition.

Effect of deformation schedule on the microstructure and mechanical properties of a thermomechanically processed C-Mn-Si transformation-induced plasticity steel

Thermomechanical processing simulations were performed using a hot-torsion machine, in order to develop a comprehensive understanding of the effect of severe deformation in the recrystallized and nonrecrystallized austenite regions on the microstructural evolution and mechanical properties of the 0.2 wt pct C-1.55 wt pct Mn-1.5 wt pct Si transformation-induced plasticity (TRIP) steel. The deformation schedule affected all constituents (polygonal ferrite, bainite in different morphologies, retained austenite, and martensite) of the multiphased TRIP steel microstructure. The complex relationships between the volume fraction of the retained austenite, the morphology and distribution of all phases present in the microstructure, and the mechanical properties of TRIP steel were revealed. The bainite morphology had a more pronounced effect on the mechanical behavior than the refinement of the microstructure. The improvement of the mechanical properties of TRIP steel was achieved by variation of the volume fraction of the retained austenite rather than the overall refinement of the microstructure.

Calcium- and vitamin D3 - fortified milk reduces bone loss at clinically relevant skeletal sites on llder men: a 2-year randomised controlled trial

In this 2-year randomized controlled study of 167 men >50 years of age, supplementation with calcium-vitamin D3-fortified milk providing an additional 1000 mg of calcium and 800 IU of vitamin D3 per day was effective for suppressing PTH and stopping or slowing bone loss at several clinically important skeletal sites at risk for fracture.

Introduction: Low dietary calcium and inadequate vitamin D stores have long been implicated in age-related bone loss and osteoporosis. The aim of this study was to assess the effects of calcium and vitamin D3 fortified milk on BMD in community living men >50 years of age.

Materials and Methods: This was a 2-year randomized controlled study in which 167 men (mean age ± SD, 61.9 ± 7.7 years) were assigned to receive either 400 ml/day of reduced fat (1%) ultra-high temperature (UHT) milk containing 1000 mg of calcium plus 800 IU of vitamin D3 or to a control group receiving no additional milk. Primary endpoints were changes in BMD, serum 25(OH)D, and PTH.

Results: One hundred forty-nine men completed the study. Baseline characteristics between the groups were not different; mean dietary calcium and serum 25(OH)D levels were 941 ± 387 mg/day and 77 ± 23 nM, respectively. After 2 years, the mean percent change in BMD was 0.9-1.6% less in the milk supplementation compared with control group at the femoral neck, total hip, and ultradistal radius (range, p < 0.08 to p < 0.001 after adjusting for covariates). There was a greater increase in lumbar spine BMD in the milk supplementation group after 12 and 18 months (0.8-1.0%, p ≤ 0.05), but the between-group difference was not significant after 2 years (0.7%; 95% CI, −0.3, 1.7). Serum 25(OH)D increased and PTH decreased in the milk supplementation relative to control group after the first year (31% and −18%, respectively; both p < 0.001), and these differences remained after 2 years. Body weight remained unchanged in both groups at the completion of the study.

Conclusions: Supplementing the diet of men >50 years of age with reduced-fat calcium- and vitamin D3-enriched milk may represent a simple, nutritionally sound and cost-effective strategy to reduce age-related bone loss at several skeletal sites at risk for fracture in the elderly.

Long-term effects of calcium-vitamin-D3-fortified milk on bone geometry and strength in older men

The long-term effects of calcium and vitamin D supplementation on bone material and structural properties in older men are not known. The aim of this study was to examine the effects of high calcium (1000 mg/day)- and vitamin-D3 (800 IU/day)-fortified milk on cortical and trabecular volumetric BMD (vBMD) and bone geometry at the axial and appendicular skeleton in men aged over 50 years. One hundred and eleven men who were part of a larger 2-year randomized controlled trial had QCT scans of the mid-femur and lumbar spine (L1–L3) to assess vBMD, bone geometry and indices of bone strength [polar moment of inertia (Ipolar)]. After 2 years, there were no significant differences between the milk supplementation and control group for the change in any mid-femur or L1–L3 bone parameters for all men aged over 50 years. However, the mid-femur skeletal responses to the fortified milk varied according to age, with a split of ≤62 versus >62 years being the most significant for discriminating the changes between the two groups. Subsequent analysis revealed that, in the older men (>62 years), the expansion in mid-femur medullary area was 2.8% (P < 0.01) less in the milk supplementation compared to control group, which helped to preserve cortical area in the milk supplementation group (between group difference 1.1%, P < 0.01). Similarly, for mid-femur cortical vBMD and Ipolar, the net loss was 2.3 and 2.8% less in the milk supplementation compared to control group (P < 0.01 and <0.001, respectively). In conclusion, calcium–vitamin-D3-fortified milk may represent an effective strategy to maintain bone strength by preventing endocortical bone loss and slowing the loss in cortical vBMD in elderly men.

The effect of Nb, Mo, and A1 additions on the transformation behaviour and mechanical properties of thermomechanically processed C-Si-Mn trip steel

The effect of additions of Nb, A1 and Mo to Fe-C-Mn-Si TRIP steels on the final microstructure and mechanical properties after simulated thermomechanical processing (TMP) has been studied. Laboratory simulations of continuous cooling during TMP were performed using a quench deformation dilatometer, while laboratory simulations of discontinuous cooling during TMP were performed using a hot rolling mill. From this a comprehensive understanding of the structural and kinetic aspects of the bainite transformation in these types of TRIP steels has been developed. All samples were characterised using optical microscopy and XRD. The relationships between the morphology of bainitic structure, volume fraction, stability of RA and mechanical properties were investigated.

Deformation behavior of nanostructured aluminum alloy processed by severe plastic deformation

Microstructure and deformation behavior of the commercial aluminum-based Al7.5%Zn–2.7%Mg–2.3%Cu–0.15%Zr alloy subjected to high pressure torsion (HPT) were studied in the present work. A small grain size less than 100 nm, high level of internal stresses and presence of second phase nanoparticles were revealed by transmission electron microscopy (TEM) and X-ray diffraction (XRD). The nanostructured alloy processed by HPT exhibits tensile strength of 800 MPa and ductility of 20% at optimal temperature-strain rate conditions. Unusual influence of a short pre-annealing on tensile strength and ductility of as-processed alloy is discussed.

Three-dimensional atom probe analysis of solute distribution in thermomechanically processed TRIP steels

Complex multiphase microstructures were obtained in transformation induced plasticity C–Mn–Si–(Nb–Al–Mo) steels by simulated controlled thermomechanical processing. These microstructures were characterized using transmission electron microscopy, X-ray diffraction and three-dimensional atom probe tomography (APT), which was used to determine the partitioning of elements between different phases and microconstituents. The measured carbon concentration (not, vert, similar0.25 at%) in the ferrite of carbide-free bainite was higher than expected from para-equilibrium between the austenite and ferrite, while the concentrations of substitutional elements were the same as in the parent austenite suggesting that incomplete bainite transformation occurred. In contrast, the distribution of substitutional elements between the ferrite lath and austenite in carbide-containing bainite indicated a complete bainite reaction. The average carbon content in the retained austenite (3.2 ± 1.6 at%) was somewhat higher than the T0 limit. On the basis of the APT measured composition, the calculated Ms temperatures for retained austenite were above room temperature, indicating its low chemical stability.

Microstructure and mechanical properties of thermomechanically processed C-Si-Mn steels

Comparison of the microstructures formed in the specimens produced by corresponding schedules in the dilatometer and by laboratory rolling has shown that a higher level of retained austenite was achieved in dilatometer specimens, whereas in rolled specimens a higher amount of martensite was present instead of retained austenite.

Functional studies on the Wilson copper P-Type ATPase and toxic milk mouse mutant

The Wilson protein (WND; ATP7B) is an essential component of copper homeostasis. Mutations in the ATP7B gene result in Wilson disease, which is characterised by hepatotoxicity and neurological disturbances. In this paper, we provide the first direct biochemical evidence that the WND protein functions as a copper-translocating P-type ATPase in mammalian cells. Importantly, we have shown that the mutation of the conserved Met1386 to Val, in the Atp7B for the mouse model of Wilson disease, toxic milk (tx), caused a loss of Cu-translocating activity. These investigations provide strong evidence that the toxic milk mouse is a valid model for Wilson disease and demonstrate a link between the loss of catalytic function of WND and the Wilson disease phenotype.

Demographic differences in usage and attitudes to milk

Declining milk consumption is a potential public health problem because milk provides nutrients that are not abundant in other foods. Identification of the factors that may influence milk consumption may lead to development of interventions to promote more healthful behaviours.

Attitudes and beliefs about food appear to be strong predictors of dietary behaviour. The objective of this study was to survey a random sample of consumers in regard to their milk consumption and attitudes and beliefs about milk. Two telephone surveys were conducted one year apart. The questionnaire included attitude items that reflected the main themes of consumers' interest in milk.

The respondents' attitudes were complex and were related to demographics and milk consumption. In general, people's concerns about milk related to what was important in their lives; what threatened them physically and emotionally. Women held more positive attitudes, but they were concerned about the fat content of milk. Men were less aware of milk's nutritional benefits and, as a result, were less appreciative of its value.

The findings from this investigation provide an opportunity to develop appropriate public health initiatives to promote the consumption of high calcium foods and to address the barriers to drinking milk. Nutrition communications in collaboration with other health agencies and the milk industry could support these initiatives.