112 research outputs found
The role of lipid peroxidation in the pathogenesis of Duchenne muscular dystrophy
No full textDuchenne muscular dystrophy (DMD) is a progressive muscle disease which is eventually fatal due usually to respiratory and cardiac failure. Although DMD is known to be an inherited, X-linked, disease the nature of the genetic defect remains elusive. Biochemical and histological evidence points to a lesion in the cell membranes of muscle and possibly other cell types although the nature of this lesion has not been elucidated. One of the many theories is that membrane lipid peroxidation causes the membrane damage and consequent necrosis seen in DMD. The aim of this work was to search for evidence relating to this theory using cultured skin fibroblasts (CSFs) and blood plasma. It was demonstrated that whole cultures of DMD CSFs, show markedly increased MDA production which distinguished them from normal control CSFs. Both CSFs grown in a medium without PUFA and with PUFA showed that lipid peroxidation occurs in a time-dependent manner and higher levels of MDA were found in control and EMD CSFs incubated in PUFA. It was also found that washed CSFs showed a considerable increase in MDA production compared with unwashed ones. The concentration of tert-butyl hydroperoxide (TBH) which is just below the minimum toxic level for CSFs was determined and found to be 50mumol/l. Although lipid peroxidation products (MDA and FP) were not significantly different in the washed particulate fraction from DMD and normal control CSFs, these products (CD, MDA and FP) were significantly higher in total homogenates from EMD compared with normal control CSFs suggesting that membrane lipid peroxidation may not be entirely membrane-dependent but also probably involves cytoplasmic components. Protection by a GSH-dependent cytosolic factor from CSFs against lipid peroxidation in a model system was examined. This work strongly indicates that DMD CSFs have a better protection system than normal controls. All three lipid peroxidation products (CD, MDA and FP) were measured in plasma and found to be significantly Increased in DMD (P0.05). A general but small decrease in alpha-tocopherol was seen in both EMD and control during storage (-20°C) up to 3 years. There was no correlation between age and vitamin E concentration for a group of adults (ages 18-35) and children (ages 3-14). Lastly plasma transferrin concentration shows no significant difference between DMD and normal controls (P>0.05) but a significant increase in caeruloplasmin was found in DMD patients compared with normal controls (P<0.001). Neither levels of transferrin nor caeruloplasmin (in IWD and normal controls) were influenced by time of storage (-20°C). Further both proteins showed no significant difference with age of subject
The Life and Letters of the Lady Arbella Stuart
No full textLady Arbella Stuart, a woman nearly forgotten in history and literature and yet a woman who lived a full and exciting life which is well documented in her letters to her family, friends and royalty (both Queen Elizabeth I and James VI and I). Arbella Stuart was born in 1575 to Elizabeth Cavendish and Charles Darnley and was brought up by her maternal grandmother, Bess of Hardwick. She was educated from birth about her proximity to the throne (there was a chance she could have been queen when Elizabeth died) and the important role she had in life. There have been several biographies written about Stuart over the years and most recently an excellent text of her existing letters by Sara Jayne Steen which is the primary source of information for this thesis. This thesis examines Stuart’s tone, rhetoric and style in a selection of letters written over the course of her life, where possible using manuscripts viewed in the British Library and Hardwick Hall, as well as the published text. Part of what makes Stuart such an interesting subject is her ability to manipulate her reader and assume different personae, depending on whom she was writing to. The young Stuart writes passionately and often without thinking first, putting her thoughts on paper and then quickly sending them off to the Queen and her advisers. An older and wiser Stuart writes from James VI and I’s court and is very formal in her letters to the King. She is more relaxed when writing to her Aunt and Uncle and depicts court life in a lively informal fashion giving us a valuable insight into what life as a courtier would have been like at this time. Finally the thesis examines Stuart’s last letters written from imprisonment, the work of a desperate woman, fighting for her freedom. Stuart, like most of us, had a multi-faceted personality. She was at times an apparently submissive and subservient subject of the King; a well read and educated woman who adopted the guise of humility and deference to those in authority, the patriarchal order in place. This thesis will depict the many different sides to Stuart and give a brief overview of her exciting and turbulent life, told through her letters
Biochemical abnormalities in erythrocytes from patients with Duchenne muscular dystrophy and multiple sclerosis
No full text(1) Duchenne Muscular Dystrophy (DHD) Muscle fibres and erythrocytes in DMD exhibit many abnormalities of me membrane-associated properties which have led to the 'Membrane Hypothesis'. The Membrane Hypothesis states that the primary lesion in DMD involves a generalized defect in the plasma membrane. Since phospholipids play a crucial part in the structure, organization and function of membranes, subtle changes in these lipids could well be responsible for the observed abnormalities. One possible alteration is a perturbation of the asymmetric distribution of phospholipids in the plasma membrane. Such asymmetry may be studied in erythrocytes using phospholipases. In this work, bee venom phospholipase A2 was used to treat intact cells and derived ghosts. Two-dimensional TLC was used to separate the extracted lipids, which were quantified by phosphorus assay. Results showed the lipid composition is normal but the degradation of PC in DMD erythrocytes was higher and the differences were highly significant (P < 0.01). Increased PC degradation in erythrocytes may be explained in at least two ways: (i) transbilayer translocation of PC occurs more readily during the course of the experiment, or (ii) more PC is localized in the outer leaflet. If the results can be explained by (i) then spectrin, which is essential in maintaining lipid asymmetry, may be of abnormal structure and in fact, abnormalities of spectrin in DMD erythrocytes have already been reported. If the explanation for the results is (ii), lipid rearrangement and changes in viscosity and fluidity can be expected, which would also result in abnormalities in spectroscopic data, osmotic fragility, ion transport and enzyme activities reported in DMD erythrocytes. Na+ -K+ ATPase activity of normal erythrocytes was reported to become 'Duchenne like' after incubation of cells with DMD plasma. A circulating factor from necrotic muscle was proposed to be responsible. The effect of DMD plasma on the asymmetry of membrane lipid was investigated but results showed that DMD plasma did not result in altered asymmetry in control cells. So the factor, if one exists, is not responsible for the increased PC degradation found in DMD erythrocytes and also the change in Na+-K+ ATPase activity is not due to a change in lipid asymmetry of the erythrocytes. (2) Multiple Sclerosis (MS) MS is a demyelinating disease of unknown cause. A dietary defect and abnormal immune response have been proposed. Lipid organization of the membrane of MS erythrocytes was investigated in this work to explore whether the reported abnormal physical properties in MS erythrocytes were due to abnormal lipid asymmetry. Results showed the membrane lipid composition and asymmetrical organization of MS is normal. So the reported increased cell size, increased osmotic fragility and reduced electrophoretic mobility of erythrocytes as well as increased platelet stickiness in MS patients is not due to changes in lipid organization in the MS erythrocyte membrane. Glutathione peroxidase, one of the enzymes which protects against membrane lipid peroxidation was reported to be decreased in MS erythrocytes. This might result in increased subsceptibility to lipid peroxidation of MS erythrocytes. The peroxidisability (using H2 O2 as stressor) and the activities of the four enzymes, glutathione peroxidase, glutathione reductase, catalase and superoxide dismutase, were investigated. Results showed the per-oxidisability of MS erythrocytes was significantly decreased (P < 0.001) which indicates that the membrane lipids of MS erythrocytes are less susceptible to peroxidation. This may be due to increased content of antioxidants (e.g. vitamin E, etc.) in the cells since the activities of the antioxidant enzymes were found to be normal in this work. Hyperbaric oxygen (HBO) treatment has been proposed for treatment of MS patients. The effect of HBO treatment on the four antioxidant enzymes were investigated. Only catalase activity was found to be increased in erythrocytes of MS patients after HBO treatment. So raised catalase may be an important effect and outweigh the potentially damaging increased lipid peroxidation which may also accompany HBO treatment
Cell wall composition and ultrastructure of the extremely halophilic coccus, sarcina marina
No full textCells of S. marina (N.C.M.B, 778) were disrupted using a Hughes press and a purified cell wall fraction obtained using a previously reported method for halococcal wall isolation. This procedure was monitored by examination of thin sections in the electron microscope and the final wall preparation was seen to be relatively free of cytoplasmic and membranous contaminants. However, treatment of the wall fraction with crude trypsin did appear to remove particulate surface components. The total ninhydrin-positive components detectable accounted, for only about 14% of the cell wall dry weight. The major amino acids present were glycine, alanine, glutamic acid and aspartic acid although very small amounts of others were detected. The amino sugar components included glucosamine and galactosamine although these only accounted for some 60% of the total amino sugars. The remainder was probably made up of one or more of four unidentified, acid-labile components detected on amino acid analysis and by paper chromatography. This is in accord with the finding of unusual, labile amino sugars in the cell walls of other halococcal species. Approximately 37% of the cell wall dry weight was made up of the neutral sugars, glucose, galactose and mannose which were present in eguimolar amounts. In addition, the wall was found to contain a negligible lipid (0.1% dry weight) and a high ash (9.2% dry weight) content. The poor recovery of organic material after analysis is almost certainly due to the lability of some of the more unusual (and in this work unidentified) components. Attempts to selectively solubilise the wall material with a view to identifying discrete polymers met with some success. In Particular, treatment with trichloroacetic acid (TCA) at 35° extracted all of the glactosamine from the wall (in addition to other components) but none of the unknown component, X1. Further treatment with TCA at 60 extracted all of another unknown component, X1. These results suggest that some degree of resolution of different polymers constituting the wall may be possible and may have been achieved here. Treatment of S. marina with the antibiotics, D-cycloserine, novobiocin, bacitracin, penicillin G and vancomycin, known to affect cell wall biosynthesis in other bacteria, was carried out. Possible effects of the antibiotics were monitored by electron microscopy and turbidimetric estimation of bacterial growth. Only novobiocin and bacitracin had any effect on growth but this was marked; in both cases growth was prevented by addition of the antibiotic. The other three antibiotics all lost their antibiotic activity (against appropriate indicator organisms) when incubated over a period of a few hours in Dundas medium. It is suggested that this may be a significant consideration when explaining the antibiotic insensitivity of microorganisms, such as S. marina whose doubling times are of the same order of magnitude as that necessary for antibiotic inactivation. Thin sections of control and antibiotic-treated cells showed interesting ultrastructural features comparable with those seen in more conventional halophilic cocci. Some minor ultrastructural changes were seen in some of the anti-biotic-treated cells, the most notable being extensive plasmolysis in the case of novobiocin. However, none of the antibiotics tested appeared to cause cell lysis or osmotic fragility which may preclude their use as agents for the non-destructive removal of the cell wall
Structural aspects of the membrane and ultrastructural features of sarcina flava and sarcina morrhuae
No full text1. The preparation, purification and properties of a water-soluble membrane component from S. flava using the synthetic detergent Lubrol L has been described. This fraction contained carotenoid, glucose and peptide, was highly stable to heat and pH extremes, and release of free carotenoid from it proved extremely difficult. The possible effect of the binding of the membrane components within detergent micelles is discussed and the dangers inherent, in the determination of molecular weights, by osmometry or ultracentrifugation, in the presence of detergent have been indicated. 2. The polar carotenoid subfractions from S. flava have been characterised and found to consist of carotenoid, glucose and peptide. The linkage between carotonoid and glucose is presumed to be glycosidic, and a model for the in vivo orientation of the carotenoid complex in the bacterial membrane has been proposed. A possible correlation between membrane stability and carotenoid content has been found, and this is discussed in relation to the model. 3. After complete removal of the free pigments from S. morrhuae by solvent extraction, a water soluble carotenoid fraction was isolated and characterised. The material, whose molecular weight is approximately 9,000 contains carotenoid, glucose and peptide. The bond between glucose and carotenoid is again presumed to be glycosidlc, and the peptide moiety contains high proportions of the acidic amino acids, the significance of which is discussed. This bound pigment is also thought to represent one form in which: carotenoid is bound in the bacterial membrane. 4. The effect of the age of the culture on the chemical composition of the total membrane fraction from S. flava has been investigated. Both protein and lipid contents decrease with age although there is little variation in carbohydrate content. It is suggested that the decreased lipid content is a reflection of the increased binding of lipid to protein with age. Considerable variation in the fatty acid composition with age was observed, which makes the use of the fatty acid profile as a taxonomic criterion for this species of doubtful value. The presence of a sterol in S. flava membrane lipids is indicated although from GLC data, it seems unlikely that this is cholesterol. Mono-saccharides detected in membrane hydrolysates were ribose, rhamnose, glucose, and mannose. The presence of glucosamine and galactosamine was also indicated. 5, The general ultrastructural features of whole cells of both S. flava and S. morrhuae have been described. S. flava exhibits many of the fine structural features common to Gram-positive organisms and was seen to form the packets of cells typical of the Sarcinae. Cell division in S. flava was shown to be of the cell membrane septation type, and a mechanism for this mode of division has been proposed. Preparation of protoplasts from S. flava using the method of Baird-Parker and Woodroffe (1967) was unsuccessful, but the treatment with lysozyme, revealed a layered appearance of the cell wall. Several intracytoplasmic membranous inclusions were seen in these cells and their relationship to mesosomes is discussed. Mesosomes as such were also present, but never in association with developing septa. The effect of varying conditions of fixation on the fine structure of S. flava was also studied. Evidence has been presented that, under certain conditions, sporulation may occur in S. flava. Good fixation of S. morrhuae cells proved difficult to achieve, and this may be due to insufficient concentration of salts in the fixation medium. Cells of S. morrhuae are approximately twice the size of S. flava cells, and division was seen to occur in a much more random fashion, producing irregular clumps of cells with common cell walls. Spherical or ovoid bodies of unknown composition were seen in association with the cytoplasmic membrane
Lipid composition and habitat selection in higher plants
No full textLipid analyses of the leaves of Empetrum nigrum subspp. hermaphroditum with an upland distribution in the U.K. and the lowland E. nigrum subspp. nigrum revealed a) that the lowland subspecies had higher total and neutral lipid levels throughout 1979 b) that total lipid levels remained constant within the leaves of both subspecies throughout the year. c) that storage lipid (triacylglycerols) contributed 1.4% and 4.5% to the total lipid of subspecies hermaphroditum and nigrum respectively. This data is inconsistent with the suggestion that the high leaf total lipid levels associated with alpine species represent high levels of storage lipid. Instead it is suggested that the high lipid content of Empetrum leaves may be a reflection of a well-developed waxy cuticle. It'is pseudacorus occupies habitats characterized by poor O2 availability and is able to tolerate up to two months total anoxia without any loss in viability. By contrast the cultivated Iris qerrnanica var Quechei typically a plant of well drained soils suffers 100% mortality during 8 weeks anoxia. Further the cut primary shoot of I. germanica was observed to be more susceptible to anoxic injury than the I. remainder of the rhizome. As the biosynthesis of polyunsaturated fatty acids requires the participation of molecular oxygen it was thought profitable to compare what changes occurred in the anoxia tolerant I. pseudacorus and intolerant I. germanica when subject to anoxic stress. In I. pseudocorus there were a number of lipid modification during anoxia. Glycolipids declined dramatically and although all fatty acids declined it was surprising that saturated acids decreased the most. It was suggested that the decline in glycolipids might reflect mobilization of carbohydrate reserves and/or a replenishment of the fatty acid pool through glycolipid breakdown. The significance of the alterations in membrane fluidity which might be expected to result from alterations in the saturated /unsaturated ratio remain unexplained. By complete contrast, the anoxia intolerant I. germanica although possessing a highly similar lipid profile exhibited no changes in lipid composition in response to anoxia. Therefore membrane dysfunction through lipid component omission is not a major factor in anoxic mortality. Through production of cytotoxic species such as H2O2, O2, OH and 1O2, O2 may bring about peroxidative damage. On rexposure to air it was found that the highly anoxia sensitive primary shoot tissue of I. germanica produced 38 times more malondialdehyde (M.D.A. - a lipid peroxidation product) than material which was maintained aerobically. 1. pseudacorus did not exhibit such differences. Although the overall levels of M.D.A. are higher in I. pseudacorus it may be that the primary shoot tissue contains efficient endogenous secondary protection mechanisms to make good peroxidative damage. However, in the natural environment it is unlikely that the species would ever be exposed to such rapid alterations in O2 concentrations
Indices of, and protective mechanisms against, lipid peroxidation in blood and cerebrospinal fluid in multiple sclerosis
No full textLipid production and composition in haploid and diploid strains of Aspergillus nidulans
No full textSix auxotrophic mutants of A. nidulans were crossed in a dialell cross system to obtain heterokaryous and heterozygous diploids. In order to ascertain their lipid accumulation ability, some of these mutants and diploids were tested in a minimal medium (with 3% glucose + 0.6% NaNO3), either in a shaker or in an incubator without agitation. Both, the mutants and diploids, exhibited only 4.6% lipid, on a dry weight basis. With the aim of optimising culture conditions for lipid accumulation, a wild type was cultivated in a range of different media and cultural conditions. The best yield (about 24%), was achieved in a modified minimal medium (MM + 12% glucose + 0.1% NaNO3), with a vortex stirrer device. The lipid composition of wild type 16 grown in a fermenter was determined. The results obtained from cells grown in two different media and using two extraction methods were compared. Fractionation of the total lipid on a Florisil column showed that this strain is composed of 86% neutral lipid, 7% glycolipid and 7% phospholipid, after isopropanol (IP) extraction, whilst chloroform-methanol (CM) extraction gave 75% neutral lipid, 8% glycolipid and 17% phospholipid. A further fractionation on hydrated Florisil showed that CM extracted sterols (both free and esterified) more efficiently than IP. Therefore, CM was considered a better extraction method, particularly for protein-bound lipids. The separation of the neutral lipid fraction into sub-classes also showed that the enhanced lipid content achieved in modified minimal medium, compared with a previously reported medium, was accounted for mainly by an increase, not in the triglycerides as was expected, but in the amount of sterols. TLC analysis of glycolipid and phospholipid from IP and CM extraction demonstrated two major glycolipid components (monoglycosyl and diglycosyl diglycerides) and that phosphatidylcholine (PC) and phosphatidylethanolamine (PE) were the principal phospholipids with lesser amounts of phosphatidylinositol, phosphatidylserine, phosphatidylglycerol, cardiolipin and phospbatidic acid (PA) after CM extraction, whilst after IP extraction only PC, PE and PA were found. Another significant difference between the two extraction methods is the large amount of PA found after CM extraction, but not after IP, showing that, almost certainly, phospholipase D activity had occurred during the process of extraction and/or storage of the lipid. It was also found that the principal phospholipid attacked by the enzyme was PC. The fatty acid composition was determined by GLC. The major fatty acids found in the total lipid were: 16:0 =21%; 17:0 =5%; 18:0 =18%; 18:1 = 20%; 18:2 = 35%. Each lipid class showed a different and distinctive fatty acid composition, exhibiting variation with the growth medium and extraction method used. Of particular interest was the sterol ester fraction which contained margarinic acid (17:0) as its only fatty acid
The Layburnes and their world, circa 1620-1720: the English Catholic community and the House of Stuart
No full textThis thesis concerns Catholics in north-western England in the late
seventeenth and early eighteenth centuries, in particular the Layburne family
of Cunswick, Cumbria. It examines their role in local society and at the courts
of the Stuart queens in London and St Germains. It traces their growing
commitment to the Jacobite cause and their hopes of thereby regaining
positions of influence at court and in the country.
The north-western Tory gentry's sympathy with their Catholic counterparts is
contrasted with the treatment given to the Quakers in the same area. The latter
were regarded as a danger to the fabric of society, representing an economic
and political threat to the government. As an example of how integrated the
Catholics were, the services in Kendal parish church were more Papist than
non-conformist, even under the Protectorate. At the Restoration the Catholics
continued to contribute to the upkeep of the church and were well-regarded in
the area.
The Layburnes occupied positions during the reign of James II, both in the
north-west and at court. Bishop John Laybume acted as James II's Catholic
bishop, and had also been involved in the Secret Treaty of Dover in 1670,
under Charles II. during James II's reign bishop Layburne had organised the
funding of Catholic chapels, clergy and education. This activity was
discovered and used in the prosecution of Catholic gentry in the trials
following the Lancashire Plot (1694). On acquittal, the Jacobites vigorously
renewed their plotting in Lancashire. Planning for a Jacobite invasion reached
its culmination in the 1715 Rising, only to end with the siege of Preston.
Despite some executions and the forfeiture of estates, many Catholic Jacobite
families survived the 1715 rising. Few rose in 1745 and many Catholic
families, with the exception of the Layburnes, prospered and continue to this
day
The manuscript miscellany in early Stuart England : a study of British Library Manuscript Additional 22601 and related texts
No full textPhDThis thesis is an intensive study of a manuscript miscellany dating from the early years
of the reign of James VI and 1: British Library Manuscript Additional 22601. Compiled
by someone who had close links to the court, but who was also likely to have been
associated with the Inns of Court and possibly with the south-west of England, the
miscellany contains verse (including that of King James) and prose in a wide range of
genres, with a particular interest in the political culture of the period. My thesis
provides a description of the manuscript's contents as a whole and then goes on to focus
on texts from three specific genres: the letter, political prose, and poetry. Studying
these individually and in their immediate context, it goes on to trace their appearance in
a number of other contemporary miscellanies held in British and North American
archives.
The two primary contentions of the thesis are (1) that manuscript miscellanies need to
be treated as coherent wholes, whose arrangement to some extent determines the
meaning of the texts they contain and (2) that in the process of transmission from one
manuscript to another texts and their meanings are significantly modified. The act of
transcription is thus also an act of interpretation. Building on work by Peter Beal, Mary
Hobbs, Harold Love, Henry Woudhuysen and others, the thesis aims to expand our
understanding both of the culture of scribal publication and of the ways in which that
culture engaged with the political, religious and literary life of the nation
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