43 research outputs found
A Quantitative Analytical Method to Test for Salt Effects on Giant Unilamellar Vesicles
Today, free-standing membranes, i.e. liposomes and vesicles, are used in a multitude of
applications, e.g. as drug delivery devices and artificial cell models. Because current
laboratory techniques do not allow handling of large sample sizes, systematic and
quantitative studies on the impact of different effectors, e.g. electrolytes, are limited.
In this work, we evaluated the Hofmeister effects of ten alkali metal halides on giant
unilamellar vesicles made of palmitoyloleoylphosphatidylcholine for a large sample size by
combining the highly parallel water-in-oil emulsion transfer vesicle preparation method with
automatic haemocytometry. We found that this new quantitative screening method is highly
reliable and consistent with previously reported results. Thus, this method may provide a
significant methodological advance in analysis of effects on free-standing model
membranes
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Oxidative discolouration in whole-head and cut lettuce: biochemical and environmental influences on a complex phenotype and potential breeding strategies to improve shelf-life
Lettuce discolouration is a key post-harvest trait. The major enzyme controlling oxidative discolouration
has long been considered to be polyphenol oxidase (PPO) however, levels of PPO and subsequent development of discolouration symptoms have not always correlated. The predominance of a latent state of the enzyme in plant tissues combined with substrate activation and contemporaneous suicide inactivation
mechanisms are considered as potential explanations for
this phenomenon. Leaf tissue physical properties have
been associated with subsequent discolouration and
these may be influenced by variation in nutrient
availability, especially excess nitrogen and head maturity at harvest. Mild calcium and irrigation stress has
also been associated with a reduction in subsequent
discolouration, although excess irrigation has been
linked to increased discolouration potentially through
leaf physical properties. These environmental factors,
including high temperature and UV light intensities,
often have impacts on levels of phenolic compounds
linking the environmental responses to the biochemistry
of the PPO pathway. Breeding strategies targeting the
PALand PPOpathway biochemistry and environmental
response genes are discussed as a more cost-effective
method of mitigating oxidative discolouration then
either modified atmosphere packaging or post-harvest
treatments, although current understanding of the
biochemistry means that such programs are likely to
be limited in nature and it is likely that they will need to be deployed alongside other methods for the foreseeable future
Autosomal dominant hypercalciuria in a mouse model due to a mutation of the epithelial calcium channel, TRPV5
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118040.pdf (publisher's version ) (Open Access)Hypercalciuria is a major cause of nephrolithiasis, and is a common and complex disorder involving genetic and environmental factors. Identification of genetic factors for monogenic forms of hypercalciuria is hampered by the limited availability of large families, and to facilitate such studies, we screened for hypercalciuria in mice from an N-ethyl-N-nitrosourea mutagenesis programme. We identified a mouse with autosomal dominant hypercalciuria (HCALC1). Linkage studies mapped the Hcalc1 locus to a 11.94 Mb region on chromosome 6 containing the transient receptor potential cation channel, subfamily V, members 5 (Trpv5) and 6 (Trpv6) genes. DNA sequence analysis of coding regions, intron-exon boundaries and promoters of Trpv5 and Trpv6 identified a novel T to C transition in codon 682 of TRPV5, mutating a conserved serine to a proline (S682P). Compared to wild-type littermates, heterozygous (Trpv5(682P/+)) and homozygous (Trpv5(682P/682P)) mutant mice had hypercalciuria, polyuria, hyperphosphaturia and a more acidic urine, and approximately 10% of males developed tubulointerstitial nephritis. Trpv5(682P/682P) mice also had normal plasma parathyroid hormone but increased 1,25-dihydroxyvitamin D(3) concentrations without increased bone resorption, consistent with a renal defect for the hypercalciuria. Expression of the S682P mutation in human embryonic kidney cells revealed that TRPV5-S682P-expressing cells had a lower baseline intracellular calcium concentration than wild-type TRPV5-expressing cells, suggesting an altered calcium permeability. Immunohistological studies revealed a selective decrease in TRPV5-expression from the renal distal convoluted tubules of Trpv5(682P/+) and Trpv5(682P/682P) mice consistent with a trafficking defect. In addition, Trpv5(682P/682P) mice had a reduction in renal expression of the intracellular calcium-binding protein, calbindin-D(28K), consistent with a specific defect in TRPV5-mediated renal calcium reabsorption. Thus, our findings indicate that the TRPV5 S682P mutant is functionally significant and study of HCALC1, a novel model for autosomal dominant hypercalciuria, may help further our understanding of renal calcium reabsorption and hypercalciuria