Carole E. Rolph

Effects of environmental factors and metals on Selenastrum capricornutum lipids

Abstract Studies of the effects of illumination, temperature and heavy metal exposure (Cu 2 , Zn 2 , Cd 2 ) on Selenastrum capricornutum have shown that these factors alter both the total fatty acid and free sterol composition of cells in batch culture. Dark treatment resulted in a decrease in the relative proportions of oleate (18 : 1) and an increase linoleate (18 : 2), together with an increase in the relative proportion of the sterol 24-ethyl-5 α -cholest-7-en-3 β -ol (Δ 7 -chondrillastenol) and a decrease in 24-ethyl-5 α -cholesta-7,22-dien-3 β -ol (chondrillasterol). A shift in temperature from 25° to 10° led to an increase in the relative proportion of oleate and a decrease in linoleate and parinate (18 : 4), together with a significant increase in the relative proportion of 24-methyl-5 α -cholest-7-en-3 β -ol (ergostenol). Generally, exposure to heavy metal ions led to an increase in oleate (with all three metals) and altered the relative proportions of linoleate and parinate (changes being metal specific). Metal (ion) treatment also significantly increase Δ 22 desaturation of the 24-ethyl sterol components. The changes in the composition of many of the individual lipid components in response to heavy metal ion exposure occurred at concentrations which did not significantly affect the organisms specific growth rate. For example, the relative proportion of oleate was affected with only 1 μ M Cu 2 in solution ( P

Streptozotocin-induced type 1 diabetes mellitus alters the morphology, secretory function and acyl lipid contents in the isolated rat parotid salivary gland

Diabetes mellitus (DM) is associated with numerous conditions including hypo-secretion of digestive enzymes. This study investigated the morphology, secretory function (α-amylase release) and acyl lipid contents in the isolated parotid gland of STZ-induced diabetic and age-matched control rats in order to provide insights into diabetes-induced salivary insufficiency. The techniques employed included light microscopy, colourimetric and gas chromatography (GC) analysis, respectively. Diabetes mellitus was induced in adult male Wistar rats by a single intraperitoneal (IP) injection of streptozotocin (STZ) (60 mg per kg body weight). Control animals were injected with a similar volume of citrate buffer. The animals were tested for DM 4 days after STZ injection and 2 months later when they were humanely killed for the experiment. The morphological results showed diabetic parotid glands to be extensively infiltrated with lipid droplets of various magnitudes, whereas glands from control animals display normal structure with the absence of lipid droplets. The analysis of parotid secretory function revealed a significant (p < 0.05) dose-dependent decrease in α-amylase release in response to noradrenaline (NA) in STZ-treated glands when compared to age-match control parotid glands. Furthermore, the levels of acyl lipids (16:0, 16:1, 18:0 and 18:1) in diabetic parotid glands was significantly (p < 0.01) reduced compared to control glands, along with a reduced ratio of 16:1/16:0. The results indicate DM can elicit changes in the morphology, secretory function and acyl fatty acid quantity in the isolated rat parotid gland. (Mol Cell Biochem 261: 175–181, 2004)

Effects of ageing on morphology, amylase release, cytosolic Ca2+ signals and acyl lipids in isolated rat parotid gland tissue.

Xerostomia (oral dryness sensation) is due to dryness of the oral cavity and it is more prevalent in the elderly. This study investigated the effect of ageing on parotid gland structure and function of control (2–6 months) and aged (12, 16–18 and 22–24 months) rats employing light microscopic, colorimetric, gas chromatographic and microspectrofluorimetric methods to investigate the morphological changes of the parotid glands, amylase release, endogenous lipid distribution and cytosolic free calcium levels, respectively. When compared to controls, age-related changes were apparent in glands obtained from rats aged 16–18 and 22–24 months, which included reduced acinar cell distribution, enlarged parotid ducts with fatty and connective tissue and mast cell infiltrations. Parotid acini from 12, 16–18 and 22–24-month-old glands showed significant (p < 0.05) age-related decreases in amylase release, compared to controls when challenged with acetylcholine (ACh). No change in basal calcium signals was observed in parotid acini from 2–6 to 16–18-month-old-animals. However, stimulation of 16–18-month-old parotid acini with 10−5M ACh resulted in a significant (p < 0.001) decrease in both peak and plateau phases of the cytosolic Ca2+ signal when compared to control. Gas chromatography of de novo and essential acyl lipids revealed no changes in the amount of either acyl lipid group in glands obtained from 2–6 to 22–24-month-old animals. Lipid analysis of phospholipid associated acyl chains showed a higher relative proportion of linoleic acid in older glands. The results reveal that ageing is associated with marked and distinct morphological changes including infiltrations of lipids and mast cells of the parotid gland and decreases in amylase release and cytosolic Ca2+ signals (Mol Cell Biochem 266: 199–208, 2004)

Nile red fluorescence screening facilitating neutral lipid phenotype determination in budding yeast, Saccharomyces cerevisiae, and the fission yeast Schizosaccharomyces pombe.

Investigation of yeast neutral lipid accumulation is important for biotechnology and also for modelling aberrant lipid metabolism in human disease. The Nile red (NR) method has been extensively utilised to determine lipid phenotypes of yeast cells via microscopic means. NR assays have been used to differentiate lipid accumulation and relative amounts of lipid in oleaginous species but have not been thoroughly validated for phenotype determination arising from genetic modification. A modified NR assay, first described by Sitepu et al. (J Microbiol Methods 91:321–328, 2012), was able to detect neutral lipid changes in Saccharomyces cerevisiae deletion mutants with sensitivity similar to more advanced methodology. We have also be able to, for the first time, successfully apply the NR assay to the well characterised fission yeast Schizosaccharomyces pombe, an increasingly important organism in biotechnology. The described NR fluorescence assay is suitable for increased throughput and rapid screening of genetically modified strains in both the biotechnology industry and for modelling ectopic lipid production for a variety of human diseases. This ultimately negates the need for labour intensive and time consuming lipid analyses of samples that may not yield a desirable lipid phenotype, whilst genetic modifications impacting significantly on the cellular lipid phenotype can be further promoted for more in depth analyses.

In vitro measurement of internal hoof strain

REASONS FOR PERFORMING STUDY Strains during stance on the hoof wall surface have been measured by a number of authors in vitro and in vivo. Histological structure and mechanical properties vary through the wall thickness (radially); radial strain measurements may therefore aid the understanding of mechanical function of the capsule and adjacent tissues. OBJECTIVES To develop instrumentation capable of measuring internal hoof strain, and to carry out a preliminary comparison of normal and laminitic hooves. METHODS Six forelimbs from 4 horses, including 2 with laminitis from the same horse, were tested using an Instron test rig designed to simulate the walk at impact, midstance and breakover. Internal strains were measured at a dorsal site using strain gauges moulded into a plug made of 007 fast-set structural adhesive. In addition, kinetic and kinematic data were collected from each specimen. RESULTS When simulating the walk, a significant (P<0.0001) increase in gradient of radial tensile strain was found in a normal hoof wall, from 5.6 +/- 73.9 microepsilon at the outer gauge to 418.5 +/- 170.6 microepsilon at the inner gauge. However, radial strains measured at the inner gauge site in limbs with laminitis were found to be significantly (P<0.0001) compressive, with values of -406.7 +/- 156.3 and -109.9 +/- 72.4 microepsilon for Specimens 1 and 2, respectively. CONCLUSIONS AND POTENTIAL RELEVANCE These preliminary data indicate that a marked redistribution may well occur in the wall of laminitic hooves. With a larger sample size, the results should have relevance to the treatment and management of laminitis.

Dielectric Characterisation of Lipid Droplet Suspensions Using the Small Perturbation Technique

This work proposes a novel approach to differentiate biological cells based upon the total concentration of lipids. Lipid accumulation within cells is significant as it serves as a marker pertaining to the metabolism and oncologic state of the cell and organism. This is accomplished through dielectric characterisation of the sample. This chapter presents a preliminary proof of concept experiment using vegetable oils and cell culture media to model lipid droplets in biological cells. The experiment indicated that solutions of numerous different lipid suspensions at different concentrations can be differentiated based upon the dielectric characteristics of the sample. The dielectric constant of vegetable oils was calculated to be between 2.9 and 3.1. The dielectric constant of the suspensions reached up to 27 at a concentration of 0.5% (v/v).

The effects of limb posture on relationships between in vitro radial hoof strain, load and joint angles

REASONS FOR PERFORMING STUDY Radial strain in normal hooves has been found to vary with strain gauge location, limb posture and sample limb but reported magnitudes were considered to be low. More accurate measurement of radial strain may enhance the understanding of hoof function. OBJECTIVES To explore in vitro radial hoof strain in relation other kinetic and kinematic variables that may be related. METHODS Five normal forelimbs were removed at the proximal articular surface of the third metacarpal bone (McIII). The limbs were loaded using a modified Instron test machine. Six calibrated infrared cameras captured movement from markers on the hoof and bone fixed markers on the second and first phalanxes and McIII, whilst radial hoof strain was measured using a calibrated instrumented plug. Change in strain, joint angle and load were found at simulated walking postures and bivariate correlations were used to compare the relationships between them. RESULTS Radial strain was moderately correlated with proximal interphalangeal joint (PIPJ) rotation (r = -0.519). Large reductions in radial strain were found in loading and midstance with 10 degrees of heel lift postures. CONCLUSIONS AND POTENTIAL RELEVANCE PIPJ rotation has previously been linked to the magnitude of deep digital flexor tendon (DDFT) loads and it is therefore suspected that these loads may have the greatest influence on radial strain magnitudes. Further investigation of radial strain is needed to describe the patterns fully during the stance phase in vivo.

Modulation of phosphatidylcholine biosynthesis in celery by exogenous fatty acids.

The effects of C16 and C18 fatty acids on the synthesis of phosphatidylcholine were studied in Apium graveolens cell suspension cultures and postmitochondrial supernatants. When cells were exposed to exogenous oleic acid, the rate of phosphatidylcholine biosynthesis increased 1.4-fold within 5 min of the addition of the fatty acid to the culture medium. The sensitivity of microsomal CTP:cholinephosphate cytidylyltransferase (EC 2.7.7.15) to saturated and unsaturated fatty acids was monitored through the addition of unesterified fatty acids to postmitochondrial supernatants. The saturated fatty acids, palmitic and stearic, appeared to have little effect on CTP:cholinephosphate cytidylyltransferase activity, whereas exposure to oleic, linoleic and cis-vaccenic acids resulted in significant increases in enzyme activity. Optimal microsomal CTP:cholinephosphate cytidylyltransferase activities were achieved by the incubation of postmitochondrial supernatants with 500 microM oleate. The exogenous fatty acids were found to be incorporated into microsomal membranes in their unesterified form. Removal of unesterified fatty acids by incubation of microsomal membranes with defatted bovine serum albumin resulted in the reduction of microsomal CTP:cholinephosphate cytidylyltransferase activity; demonstrating that the enzyme requires unesterified unsaturated fatty acids.

Promising near-infrared non-targeted probes: benzothiazole heptamethine cyanine dyes

A series of benzothiazole heptamethine cyanine dyes have been synthesized and their photophysical properties evaluated in relation to their structural features. These have been compared against two classical probes of this type: Indocyanine Green (IGC) and New Indocyanine Green (IR-820). Growth inhibitory studies were also performed using a eukaryotic, unicellular organism, fission yeast Schizosaccharomyces pombe. Herein we highlight some potentially interesting candidates with improved fluorescence quantum yields when compared with ICG and IR-820. GRAPHICAL ABSTRACT

PP75. DROSOPHILA MELANOGASTER AS A MODEL ORGANISM TO STUDY GLIOMA: LOCATION AND IDENTIFICATION OF FOLLISTATIN

AbstractGliomas are the most common, malignant brain tumour found in the central nervous system. The main characteristics of gliomas is their rapid proliferation, resistance to current chemotherapies and their ability to infiltrate the brain. Brain tumours have a low survival rate due to the lack of available treatments. In the UK between 2010 and 2011, the survival rate was 14% for 10 or more year. The mortality rates for malignant brain and CNS tumours have increased by up to 24% in the UK between 1970 and 2012. As there is difficulty obtaining glioma samples, it is important to identify a suitable model organism. The main signalling pathways seen to be activated in human glioma development are the epithelial growth factor receptor (EGFR) and phosphatidylinositol-3 kinase (PI3K) pathways. The homologous EFGR and PI3K pathways have been studied greatly in drosophila, and show that there must be co-activation of the EGFR and PI3K pathways for cancer development to occur; leading to an increase in glial cell numbers by 50-100x fold. Follistatin is a protein which has been identified as a biomarker in malignant gliomas; by comparing normal serum to glioma serum it has shown that there is an overexpression of follistatin. Gliomas can be replicated in drosophila as there has been a high level of homology identified in human diseased states and in neural development. Follistatin studies have shown it has important roles in development and differentiation of tissues and organs. It acts as an inhibitor, binding to activin, bone morphogenetic proteins and other proteins which can be found in vertebrates. Follistatin has been identified within a number of different cancer types (such as prostate and ovarian), and plays an important role in cancer metastasis and angiogenesis. Serum studies have revealed follistatin is present in the early stages of cancer development; however the location and role of follistatin in cancer development has not been identified. Drosophila contain a homolog of follistatin, which will allow studies of follistatin in cancer within drosophila. By using drosophila as a model organism it will allow the location of follistatin and its involvement in glioma development to be identified. Immunohistochemistry is used to identify the location and distribution of a protein within a tissue or an organ. It is commonly used to identify which proteins are present in specific types of cancer. Follistatin has been identified to play a role in the early stages of glioma development. Therefore by using immunohistochemistry follistatin can be identified throughout the different stages of glioma development and possibly identify its role in this development. The aim of the research is to identify and locate the protein follistatin in drosophila by using immunohistochemistry. Using different larval instar stages, this will identify the distribution of follistatin in different stages of neural development. The hypothesis would be that follistatin is involved in the early stages of glial development and the distribution is important in glioma development compared to normal neural development.