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Biomedical subjects

G W Griffiths

Publications and source records attributed to G W Griffiths.

12 recordsLinked to original sources
PubMed1980

Attempts to quantitate immunocytochemistry at the electron microscope level.

The ability to localize intracellular macromolecules in situ by high resolution techniques has been made possible by the development of antibody labelling of thin sections obtained either from tissues embedded in an hydrophilic matrix, or by ultracryotomy or from conventional plastic embedded tissue. When particle-tagged immunological reagents are used to visualize intracellular antigens, quantitative information can be obtained by combining particle counts with morphometric estimations of compartment volume. Various detection systems have been used successfully for quantitation, which include ferritin-conjugated antibodies, biotin-avidin-ferritin complexes and, more recently, gold-protein A conjugates. Examples of the use of these techniques the localization of secretory proteins in pancreatic exocrine cells, opsin and a large membrane protein in photoreceptor cells of frog retina, and contractile proteins in skeletal muscle are given. Quantitative data obtained by morphometric analysis, both in bovine and rat pancreatic exocrine cells, are compared with values assessed by biochemical methods.

Amylases↗
PubMed1980

Antibody labeling of thin sections of skeletal muscle with specific antibodies: a comparison of bovine serum albumin (BSA) embedding and ultracryomicrotomy.

Bovine serum albumin (BSA) embedding and ultracryomicrotomy were used to prepare thin sections of glutaraldehyde-fixed skeletal muscle; these were then treated with antibodies against alpha-actinin, myosin, and actin. Three criteria were then used to compare these two techniques: 1) The preservation of fine structure; 2) the specificity of labeling with antibodies and 3) the amount of antibody bound to a particular antigen. Fine structure was better preserved using ultracryomicrotomy. Both techniques, under optimal conditions, gave specific labeling of muscle components. The amount of antibody bound was higher for BSA sections than for frozen sections. The conclusion is that, while ultracryomicrotomy gives superior qualitative results, the most reliable quantitative estimates would be obtained by using both methods together. Ultracryomicrotomy has the additional advantage that semithin sections can be visualized by immunofluorescence.

Actinin↗
PubMed1979

Transport of glial cell acid phosphatase by endoplasmic reticulum into damaged axons.

Earlier observations indicated that cutting the photoreceptor (R) cell perikarya in the retina of the flies Musca domestica and Calliphora erythrocephala resulted in an extremely rapid degeneration of the R-cell axons. The process manifested itself within minutes and secondary lysosomes appeared early on in the degenerating axons. In this study, biochemical assays of the classical lysosomal marker enzyme acid phosphatase (AcPase) showed a rapid increase in activity upon cutting the retina, reaching a peak around 5 min, followed by a drop and a subsequent slow steady rise in activity between about 4 and 24 h after the cut. Inhibitor studies indicated that at least 2 different acid phosphatases were involved, which showed identical activity changes. EM cytochemical studies with either beta-glycerophosphate or cytidine monophosphate as substrates indicated that the degeneration-dependent AcPase activity originated exclusively in one specific glial cell type in the distal lamina, the satellite glia. These cells are contiguous with R-cell axons in the pseudocartridges, directly proximal to the basement membrane of the retina. In normal uncut flies, reaction product indicative of AcPase activity was found in satellite glial cells in both rough ER (and perinuclear cisternae) and in smooth ER, the 2 types of cisternae being clearly in continuity. The electron-dense reaction product resulting from AcPase activity often showed continuity between the smooth ER of satellite glial cells and extracellular sites between the satellite glia and R-axons. This may result from the low levels of AcPase being synthesized in rough ER, and then exported to extra-cellular sites. Within minutes of cutting the retina the freqeuncy of satellite glia smooth ER staining for AcPase increases significantly. Increases in AcPase activity were subsequently found sequentially in extracellular sites, in axons in the distal lamina, and finally in the axon terminals. The time course for these increases in AcPase activity correlated well with the time course of the fine-structural pathological changes in the axons. A significant feature of the satellite glia cells is the massive packing of unidirectional microtubules (MTs), running perpendicular to the projections of the R-axons--in the same direction as AcPase transport. The MTs terminate at the glial membrane directly adjacent to the axon. The smooth ER cisternae interdigitate among the MTs and may be structurally connected to them by thin fibrils. Thick-section cytochemistry and stereology were used to reveal the 3-dimensional extent of the AcPase-reactive smooth ER system. Evidence is presented which suggests that this system represents GERL, a specialized region of smooth ER involved in lysosome formation, that in this glial cell appears to function also to export hydrolytic enzymes.

Acid Phosphatase↗
PubMed1977

The procoagulant activity of human granulocytes, lymphocytes and monocytes stimulated by endotoxin. Coagulation and electron microscopic studies.

Leukocytes from donor blood were separated by ficoll/Urovison density centrifugation into granulocytes, lymphocytes and monocytes. The cell fractions were suspended in a culture medium to which endotoxin of Salmonella enteritidis was added at a final concentration of 10 microgram/ml. Endotoxin-stimulated monocytes developed a very high tissue factor (thromboplastin) activity while in granulocytes an only negligible amount of tissue factor activity was detectable. The tissue factor activity measured in the preparation of the lymphocytes can be explained by contamination with monocytes. Eelectron microscopic studies showed the lysosomes of all monocytes to be enlarged and activated. Only a fraction of the granulocytes appeared degranulated with prominent vacuoles containing inclusion bodies. Possibly the high tissue factor activity of the monocytes triggers the development of the disseminated intravascular coagulation in the Shwartzman phenomenon.

Blood Coagulation Tests↗
PubMed1977

In vivo sterol biosynthesis by pea aphid symbiotes as determined by digitonin and electron microscopic autoradiography.

Pea aphid primary symbiotes have previously been shown to synthesize cholesterol in vitro. Two electron microscopic techniques were used here to determine whether the symbiotes also synthesize cholesterol in vivo and whether this cholesterol is made available to the aphid. We also inquired into a possible role of secondary symbiotes in chosesterol biosynthesis. Treatment of aphids with digitonin resulted in significant alteration of ultrastructural sites in primary and secondary symbiote membranes. We concluded that these sites are areas of high cholesterol concentration in the symbiotes. Electron microscopic autoradiography with 3H-mevalonate precursor indicated that both primary and secondary symbiotes synthesize cholesterol; in both cases, the majority of grains were associated with the symbiote membranes. While the frequency of grains on the symbiotes remained constant, irrespective of incubation time in labelled media, the frequency of grains over surrounding tissues increased exponentially as the time of incubation was increased from 30 min to 8 h, indicating that symbiote cholesterol is transported to other tissues. High voltage electron microscopic autoradiography permitted thick section autoradiography, reducing the time of emulsion exposure from 54 days (thin section) to 12 days (0.5 mum sections).

Animals↗
PubMed1977

Effect of dietary cholesterol on the pattern of osmium deposition in the symbiote-containing cells of the pea aphid.

Pea aphids left for 48 h in unbuffered osmium tetroxide show heavy staining of many organelles in the symbiote-containing cells (mycetocytes and sheath), embryos and oenocytes very similar to that characteristic of mammalian sterol-synthesizing cells. However, the staining of the per-aphid cells is, to a large extent, dependent on the presence of cholesterol benzoate, or free cholesterol, in the aphid's diet. In aphids cultured in vitro with 3H mevalonate in the presence of added cholesterol, the incorporation of label into the cholesterol and lanosterol fractions is significantly reduced. If the dietary cholesterol effects a similar inhibition in vivo, the cholesterol-dependent osmium staining could be due to precursors(s) of cholesterol accumulating in the intracellular sites described. There is also osmium staining of large (normally electron-transparent) vacuoles in mycetocytes, gut and fat body, irrespective of dietary cholesterol.

Animals↗
PubMed1975

Ultrastructure of pea aphid mycetocytes: evidence for symbiote secretion.

A detailed investigation into the ultrastructure of the pea aphid mycetocytes and their contained symbiotes and organelles was carried out with the transmission electron microscope. The most striking observation was the presence of small vesicles in the space between the primary symbiote cell wall and membrane envelope (outer membrane space). The vesicles appear to form by a budding process at the outer cell wall layer. Subsequently, the vesicles, we suggest, may move out into the mycetocyte cytoplasm via a similar budding of the membrane envelope; The Golgi apparatus was found to be an important structural component of the primary mycetocyte; it is continuous with the rough endoplasmic reticulum and the latter, in turn, appears to be closely connected to the primary symbiote membrane envelope. This may be of functional significance. A number of other organelles not previously described in mycetocytes were found, including transparent vacuoles, granular bodies, multi-vesicular bodies and microfilaments. The chemical composition of the various vesicles and organelles is unknown at present.

Animals↗