Cholesterol is necessary both for the toxic effect of Abeta peptides on vascular smooth muscle cells and for Abeta binding to vascular smooth muscle cell membranes

Accumulation of beta amyloid (Aβ) in the brain is central to the pathogenesis of Alzheimer's disease. Aβ can bind to membrane lipids and this binding may have detrimental effects on cell function. In this study, surface plasmon resonance technology was used to study Aβ binding to membranes. Aβ peptides bound to synthetic lipid mixtures and to an intact plasma membrane preparation isolated from vascular smooth muscle cells. Aβ peptides were also toxic to vascular smooth muscle cells. There was a good correlation between the toxic effect of Aβ peptides and their membrane binding. 'Ageing' the Aβ peptides by incubation for 5 days increased the proportion of oligomeric species, and also increased toxicity and the amount of binding to lipids. The toxicities of various Aβ analogs correlated with their lipid binding. Significantly, binding was influenced by the concentration of cholesterol in the lipid mixture. Reduction of cholesterol in vascular smooth muscle cells not only reduced the binding of Aβ to purified plasma membrane preparations but also reduced Aβ toxicity. The results support the view that Aβ toxicity is a direct consequence of binding to lipids in the membrane. Reduction of membrane cholesterol using cholesterol-lowering drugs may be of therapeutic benefit because it reduces Aβ-membrane binding.

A non-radioactive method for measuring Cu uptake in HepG2 cells

At present, all data on Cu uptake and metabolism have been derived from radioactive uptake experiments. These experiments are limited by the availability of the radioactive isotopes ⁶⁴Cu or ⁶⁷Cu, and their short half-life (12.5 and 62 h, respectively). In this paper, we investigate an alternative method to study the uptake of Cu with natural isotopes in HepG2 cells, a liver cell line used extensively to study Cu metabolism. In nature, Cu occurs as two stable isotopes, ⁶³Cu and ⁶⁵Cu (⁶³Cu/⁶⁵Cu = 2.23). This ratio can be measured accurately using inductively coupled plasma mass spectrometry (ICP-MS). In initial experiments, we attempted to measure the time course of Cu uptake using ⁶⁵Cu. The change in the ⁶³Cu/⁶⁵Cu ratio, however, was too small to allow measurement of Cu uptake by the cells. To overcome this difficulty, the natural ⁶³Cu/⁶⁵Cu ratio in HepG2 cells was altered using long-term incubation with ⁶³Cu. This had a significant effect on Cu concentration in HepG2 cells, changing it from 81.9 ± 9.46 pmol μg DNA⁻¹ (week 1) to 155 ± 8.63 pmol μg DNA⁻¹ (week 2) and stabilising at 171 ± 4.82 pmol μg DNA⁻¹ (week 3). After three weeks of culture with 2 μM ⁶³Cu the ⁶³Cu/⁶⁵Cu changed from 2.18 ± 0.05 to 15.3 ± 1.01. Cu uptake was then investigated as before using ⁶⁵Cu. Uptake was linear over 60 min, temperature dependent and consistent with previous kinetics data. These observations suggest that stable isotope ICP-MS provides an alternative technique for the study of Cu uptake by HepG2 cells.

Sperm cells of the pollen tubes of Brassica : ultrastructure and isolation

Sperm cells of pollen tubes grown both in vivo and in vitro form a male germ unit. Extensions from both sperm cells of each pollen tube are closely associated with the tube nucleus. A high yield (2.7 × 104. 20 mg−1 pollen grains germinated) of intact sperm cells was obtained following release by osmotic shock from pollen tubes grown in vitro. Structural integrity of isolated sperm was maintained by isolation at low temperature in an osmotically balanced medium. At 4° C many isolated sperm pairs were still enclosed within the pollentube inner plasma membrane. Sperm cells not enclosed within this membrane no longer remained connected as a pair. During isolation vesicles formed on the sperm cell surface from disruption of the fibrillar components bridging the periplasmic space. Both in the pollen tube and after isolation the sperm nucleus is in close association with at least one region of the sperm plasma membrane. Sperm isolated at room temperature showed the presence of nucleopores, and nuclei were euchromatic, instead of heterochromatic as in intact sperm in the pollen tube.

c-Cbl facilitates endocytosis and lysosomal degradation of cystic fibrosis transmembrane conductance regulator in human airway epithelial cells

Cystic fibrosis transmembrane conductance regulator (CFTR) is a cAMP-activated Cl− channel expressed in the apical membrane of fluid-transporting epithelia. The apical membrane density of CFTR channels is determined, in part, by endocytosis and the postendocytic sorting of CFTR for lysosomal degradation or recycling to the plasma membrane. Although previous studies suggested that ubiquitination plays a role in the postendocytic sorting of CFTR, the specific ubiquitin ligases are unknown. c-Cbl is a multifunctional molecule with ubiquitin ligase activity and a protein adaptor function. c-Cbl co-immunoprecipitated with CFTR in primary differentiated human bronchial epithelial cells and in cultured human airway cells. Small interfering RNA-mediated silencing of c-Cbl increased CFTR expression in the plasma membrane by inhibiting CFTR endocytosis and increased CFTR-mediated Cl− currents. Silencing c-Cbl did not change the expression of the ubiquitinated fraction of plasma membrane CFTR. Moreover, the c-Cbl mutant with impaired ubiquitin ligase activity (FLAG-70Z-Cbl) did not affect the plasma membrane expression or the endocytosis of CFTR. In contrast, the c-Cbl mutant with the truncated C-terminal region (FLAG-Cbl-480), responsible for protein adaptor function, had a dominant interfering effect on the endocytosis and plasma membrane expression of CFTR. Moreover, CFTR and c-Cbl co-localized and co-immunoprecipitated in early endosomes, and silencing c-Cbl reduced the amount of ubiquitinated CFTR in early endosomes. In summary, our data demonstrate that in human airway epithelial cells, c-Cbl regulates CFTR by two mechanisms: first by acting as an adaptor protein and facilitating CFTR endocytosis by a ubiquitin-independent mechanism, and second by ubiquitinating CFTR in early endosomes and thereby facilitating the lysosomal degradation of CFTR.

Investigational agents that protect pancreatic islet β-cells from failure

Type 2 diabetes is associated with insulin resistance and reduced insulin secretion, which results in hyperglycaemia. This can then lead to diabetic complications such as retinopathy, neuropathy, nephropathy and cardiovascular disease. Although insulin resistance may be present earlier in the progression of the disease, it is now generally accepted that it is the deterioration in insulin-secretory function that leads to hyperglycaemia. This reduction in insulin secretion in Type 2 diabetes is due to both islet β-cell dysfunction and death. Therefore, interventions that maintain the normal function and protect the pancreatic islet β-cells from death are crucial in the treatment of Type 2 diabetes so that plasma glucose levels may be maintained within the normal range. Recently, a number of compounds have been shown to protect β-cells from failure. This review examines the evidence that the existing therapies for Type 2 diabetes that were developed to lower plasma glucose (metformin) or improve insulin sensitivity (thiazolidinediones) may also have islet-protective function. Newer emerging therapeutic agents that are designed to increase the levels of glucagon-like peptide-1 not only stimulate insulin secretion but also appear to increase islet β-cell mass. Evidence will also be presented that the future of drug therapy designed to prevent β-cell failure should target the formation of advanced glycation end products and alleviate oxidative and endoplasmic reticulum stress.

One step multifunctional micropatterning of surfaces using asymmetric glow discharge plasma polymerization

Micropatterning of surfaces with varying chemical, physical and topographical properties usually requires a number of fabrication steps. Herein, we describe a micropatterning technique based on plasma enhanced chemical vapour deposition (PECVD) that deposits both protein resistant and protein repellent surface chemistries in a single step. The resulting multifunctional, selective surface chemistries are capable of spatially controlled protein adhesion, geometric confinement of cells and the site specific confinement of enzyme mediated peptide self-assembly.

hZip1 (hSLC39A1) regulates zinc homoeostasis in gut epithelial cells

Zinc is an essential trace element required for enzyme catalysis, gene regulation and signal transduction. Zinc absorption takes place in the small intestine, however, the mechanisms by which cells accumulate zinc are not entirely clear. Zip1 (SLC39A1) is a predicted transmembrane protein that is postulated, but not conclusively proven to mediate zinc influx in gut cells. The aim of this study was to investigate a role for hZip1 in mediating zinc uptake in human enterocytes. Both hZip1 mRNA and protein were detected in human intestinal tissue. In non-differentiated Caco-2 human gut cells, hZip1 was partially localised to the endoplasmic reticulum. In contrast, in differentiated Caco-2 cells cultured in extracellular matrix, the hZip1 protein was located in proximity to the apical microvilli. Lack of surface antibody binding and internalisation indicated that hZip1 was not present on the plasma membrane. Functional studies to establish a role for hZip1 in cellular zinc accumulation were carried out using 65Zn. In Caco-2 cells harbouring an hZip1 overexpression construct, cellular zinc accumulation was enhanced relative to the control. Conversely, Caco-2 cells with an hZip1 siRNA construct showed reduced zinc accumulation. In summary, we show that the Caco-2 cell differentiation endorses targeting of hZip1 to a region near the apical domain. Given the absence of hZip1 at the apical plasma membrane, we propose that hZip1 may act as an intracellular sensor to regulate zinc homoeostasis in human gut cells.

Fabrication of boron nitride nanotube-gold nanoparticle hybrids using pulsed plasma in liquid

Plasma, generated in liquid at atmospheric pressure by a nanosecond pulsed voltage, was used to fabricate hybrid structures from boron nitride nanotubes and gold nanoparticles in deionized water. The pH was greatly reduced, conductivity was significantly increased, and concentrations of reactive oxygen and nitrogen species in the water were increased by the plasma treatment. The treatment reduced the length of the nanotubes, giving more individual cuplike structures, and introduced functional groups onto the surface. Gold nanoparticles were successively assembled onto the functionalized surfaces. The reactive species from the liquid plasma along with the nanosecond pulsed electric field seem to play a role in the shortening and functionalization of the nanotubes and the assembly of gold nanoparticles. The potential for targeted drug delivery was tested in a preliminary investigation using doxorubicin-loaded plasma-treated nanotubes which were effective at killing ∼99% of prostate cancer cells.

Randomized controlled trial examining the effects of fish oil and multivitamin supplementation on the incorporation of n-3 and n-6 fatty acids into red blood cells

The present randomized, placebo-controlled, double-blind, parallel-groups clinical trial examined the effects of fish oil and multivitamin supplementation on the incorporation of n-3 and n-6 fatty acids into red blood cells. Healthy adult humans (n = 160) were randomized to receive 6 g of fish oil, 6 g of fish oil plus a multivitamin, 3 g of fish oil plus a multivitamin or a placebo daily for 16 weeks. Treatment with 6 g of fish oil, with or without a daily multivitamin, led to higher eicosapentaenoic acid (EPA) composition at endpoint. Docosahexaenoic acid (DHA) composition was unchanged following treatment. The long chain LC n-3 PUFA index was only higher, compared to placebo, in the group receiving the combination of 6 g of fish oil and the multivitamin. Analysis by gender revealed that all treatments increased EPA incorporation in females while, in males, EPA was only significantly increased by the 6 g fish oil multivitamin combination. There was considerable individual variability in the red blood cell incorporation of EPA and DHA at endpoint. Gender contributed to a large proportion of this variability with females generally showing higher LC n-3 PUFA composition at endpoint. In conclusion, the incorporation of LC n-3 PUFA into red blood cells was influenced by dosage, the concurrent intake of vitamin/minerals and gender.

Synchronization by low-amplitude light-dark cycles of 24-hour pineal and plasma melatonin rhythms of hatchling European starlings (Sturnus vulgaris)

In young European starlings, as in other avian species, high-amplitude 24-hr rhythms in plasma and pineal melatonin are already present around the time of hatching. In chickens this rhythmicity results at least partly from the light sensitivity of the melatonin-producing and -secreting system. In contrast to the chicken, the starling is a hole-nesting bird, and it seemed questionable whether the low light intensities in the nest are sufficient to synchronize perinatal melatonin rhythms. We therefore exposed starling eggs to light cycles roughly simulating those measured in nest-boxes, i.e., an 11-hr phase of complete darkness and a 13-hr phase consisting of 15 min of dim light (10 lux) alternating with 30 min of darkness. For one group the photophase lasted from 0600 to 1900 hr; for the other group the photophase lasted from 1800 to 0700 hr. In approximately 10-hr-old hatchlings of both groups, plasma and pineal melatonin concentrations were high during the dark phase and low during the light phase. We conclude that perinatal low-amplitude light intensity changes of the kind experienced by hatching starlings in the field are sufficient for synchronizing the melatonin-producing and -secreting system in the pineal and possibly other organs.

Vitamins D and A can be successfully measured by LC-MS/MS in cord blood diluted plasma

OBJECTIVES: In widely used protocols for the collection and isolation of cord blood mononuclear cells, investigators are left with substantial volumes of diluted plasma which could be used for other measurements. The aim of this study was to ascertain the validity of umbilical cord blood (UCB) diluted plasma samples for vitamin D, A and E analysis compared to UCB serum samples. DESIGN & METHODS: Twenty UCB matched samples of diluted plasma and serum were collected. The samples were analysed by two liquid chromatography-tandem mass spectrometry (LC-MS/MS) methods on two separate occasions. RESULTS: The results of 25(OH)D3 obtained by the two laboratories demonstrated close agreement with a mean difference of 0.14nmol/L [95% confidence interval (95% CI), -6.8 to 7.1]. Both methods demonstrate close agreement for 25(OH)D3 in UCB serum versus diluted UCB plasma; mean difference 2.2nmol/L [95% CI, -9.5 to 13.9] and 4.1nmol/L [95% CI, -14.5 to 6.1] for the results from Lab A and Lab B, respectively. Vitamin A was quantified by Lab A in UCB serum and diluted UCB plasma; mean difference 0.07μmol/L [95% CI, -0.41 to 0.28]. Results of 25(OH)D3 epimer and vitamin E in the diluted UCB plasma were below the limit of quantification, and could not be compared with UCB serum. CONCLUSIONS: Diluted UCB plasma can be used for the quantification of retinol and 25(OH)D3 by LC-MS/MS. By contrast, quantification of 25(OH)D3 epimer and vitamin E in diluted UCB plasma is not supported by this study due to limitations in analytical sensitivity.