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Quantification of neuropeptides in the central nervous system of the wobbler mouse during the progression of the motor neuron disease: a study by radioimmunoassay andimmunocytochemistry翁建霖, Yung, Kin-lam, Ken. January 1992 (has links)
published_or_final_version / Anatomy / Master / Master of Philosophy
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Neutrophil tissue inhibitor of matrix metalloproteinases-1 (TIMP- 1) : novel localisation, mobilisation and possible role.Price, Brendon. 15 November 2013 (has links)
At the beginning of this study, the granule localisation and regulation of release of human
neutrophil (PMNL) precursor collagenases, proMMP-8 and -9 (type I and type TV/V
collagenases, respectively), enzymes highly active against the extracellular matrix (ECM) and
thought to be relevant in invasion and inflammation, had been established while that of their
inhibitor, tissue inhibitor of matrix metalloproteinases-I (TIMP-1), had not.
Electron microscopy immunogold labelling of cryoultramicrotomy sections for granule marker
proteins, lysosome-associated membrane proteins (LAMPs) and endocytosed bovine serum
albumin-coated gold probes, followed by stereology, established that TIMP-1 was mainly
located in a distinct oval, electron translucent organelle, a little larger than azurophil granules.
A lack of labelling for endocytic markers and for glycosylphosphatidylinositol-anchored
proteins, established using granule fractionation and immunolabelling to be markers for the
secretory vesicles, and LAMPs-1 and -2, indicated the non-endosomal, non-secretory and nonlysosomal
nature of this organelle. Density gradient cofractionation with the least dense
secretory vesicle population and some pleiomorphism of the organelle suggested that it is a
"vesicle" rather than a "granule" population. Colocalisation with proMMP-9 in minor
subpopulations suggests that TIMP-1 vesicle biogenesis occurs between metamyelocytic and
termination differentiation, but before secretory vesicle synthesis.
Immunolabelling of phagocytosed and pulse-chased IgG-opsonised latex beads showed that
specific and azurophil granules and a small number of proMMP-8-containing granules (a
specific granule subpopulation) fuse with the phagosome whereas the TIMP-1 vesicle and
proMMP-9-containing granules do not, suggesting that the latter play no role in phagosomal
destruction of IgG-opsonised bacteria and that their phagosomal release is not calcium
regulated. However, studies using the calcium ionophore, ionomycin, and monitoring
extracellular granule marker protein release upon addition of increasing levels of extracellular
calcium, showed that all granules, except the TIMP-1 vesicle, appeared to be calcium regulated.
This suggests that the regulation of proMMP-9 release is not exclusively via calcium and that
TIMP-1 vesicle release is not calcium regulated. Whereas most granules were shown to be associated with microtubule-like structures, the
TIMP-1 vesicle and proMMP-9-containing granules were shown to associate with two
morphologically different cytoskeletal elements, neither resembling actin nor tubulin. These
elements, and the release of the TIMP-1 vesicle and proMMP-9-containing granules, need to be
studied further, but results achieved to date may explain the observed differential mode of
release of TIMP-1 relative to proMMP-9.
The proMMP-9-binding and inhibitory capacity of a 66 kDa high molecular mass form of
TIMP-1 was demonstrated in PMNL homogenates and plasma using western ligand blots and a
novel reverse zymography method. The role and relevance of this form remains unknown as
does the relevance and potential role of proMMP-9ffIMP-1 complexes seen during isolation
procedures. The proMMP-9ffIMP-1 complex may occur in vivo, as evidenced by
immunolocalisation studies, and, together with TIMP-1 released from its own discrete vesicle
population, may be responsible for the fine regulation of extracellular proteolysis. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 2002.
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A study of proteinases of invasive cells using cryoultramicrotomy and immunogold labelling.Elliott, Edith. January 1993 (has links)
This study forms part of an investigation into the possible relevance of the
lysosomal proteinases, cathepsins B, H, Land D, in cancer cell invasion. In this
study, the main technique adopted was the Tokuyasu "cryo" method, in which the
tissues were fixed, frozen and sectioned and labelled using the relevant antibodies,
which were detected with protein A gold probes.
In order to implement the Tokuyasu technique, it was necessary to rebuild a knife
maker, for the production of adequately sharp glass knives, and to modify a sputter-coater
into a glow-discharger, for rendering carbon-coated grids hydrophilic, to
promote adhesion of hydrated sections.
This study was directed towards human tissues and peptide antibodies were
investigated as a means of avoiding isolation of proteins from scarce human tissue,
and as a means of obtaining antibodies that will target specific regions of proteins of
interest. Peptide antibodies were also considered promising for studies of
proteinase trafficking and as immunoinhibiting agents, potentially useful in cancer
therapy. Various prediction programmes were investigated for their effectiveness
in predicting whether a given peptide sequence will elicit antibodies that will react
with the native protein. Successful prediction would increase the success rate of
peptide antibody production and thus lower the cost.
Leucocytes were studied as a model of an invasive cell, since they are more readily
available than tumour cells and serve the purpose during the development of
methods. In the course of these studies, an optimal protocol for the fixation of
PMNs was developed, involving lateral fixation of cut sections, that should be
useful for future studies on these cells. Elastase and cathepsins D and G were found
on the surface of activated PMNs and could thus play a role in the invasive
properties of these cells.
Studies on MCF-10A "normal" breast epithelial cells and their ras-transformed
Neo-T counterparts revealed that upon transformation, lysosomes shift from a
perinuclear position, to a more peripheral position. None of the cathepsins studied
was found on the cell surface of either the normal or ras-transfected cells,
suggesting that surface distribution of these enzymes may not be a requirement for
invasiveness. These studies suggest that immunocytochemical investigation of
cells, in the process of invading through a barrier membrane, might be profitable in
elucidating the role of proteinases in invasive cancer. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1993.
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