Biomedical subjects
Publications and source records attributed to J E Hesketh.
After treatment of synaptosomes with Nonidet-containing buffers, a proportion of the proteins remained insoluble. The major component (50%) of the residue was identified as a spectrin-like protein by immunodetection after mono- and bi-dimensional gel electrophoresis and transfer to nitrocellulose paper. Actin was also present.
1. Zinc binding to tubulin was studied by incubating microtubules with 65Zn and measuring the 65Zn retained with tubulin on DEAE Sephadex. 2. Data showed two classes of binding site, one of very low affinity and a second class binding 0.86 g-atoms Zn/mol tubulin with a dissociation constant of approx. 110 microM. 3. Binding of zinc to the higher affinity site was 50% inhibited by 2 mM N-ethyl maleimide.
The lability of tubulin in supernatant fluids from rat brain was studied by measuring the loss of colchicine-binding activity. During incubation at 37 degrees C for 1 hr approximately 50% of the binding activity of rat brain supernatant fluids was lost. This loss was reduced by mercaptoethanol or reduced glutathione. Anaerobic conditions did not affect loss of binding, while oxygen markedly reduced it, especially in the presence of reduced glutathione. Supernatant fluids prepared from brains of animals deficient in selenium showed a lower loss of binding than those from selenium-supplemented controls.
Male weanling rats were maintained on diets either deficient or adequate in zinc for a period of 4 weeks. The rats on the deficient diet showed a reduction in food intakes and growth. After 4 weeks both soleus muscles and the lateral portion of the diaphragm were studied histochemically to examine the relative frequencies of the fibre types. The soleus muscles of the deficient animals showed a significant change in the proportion of slow and fast fibres. The diaphragm muscles of the deficient animals had a significant increase in the proportion of fast-twitch oxidative glycolytic fibres and a significant decrease in fast-twitch glycolytic fibres compared with the controls. Stainable lipid increased in the diaphragm muscle of the deficient animals with respect to their pair-fed controls.
1. Microtubule reassembly was studied in supernatant fluids from rat brain. Tubulin in extracts from zinc-deficient animals showed an impaired ability to repolymerize compared to extracts from controls; 10 microM zinc stimulated reassembly of tubulin in extracts from zinc-deficient animals. 2. Low zinc concentrations (250-900 microM zinc in the presence of 1 mM EGTA) stimulated reassembly of tubulin in brain extracts from control rats; similar concentrations of nickel had no effect whilst cobalt was inhibitory. In the absence of EGTA 20-40 microM zinc stimulated reassembly in brain extracts from normal rats. 3. Zinc-induced changes in reassembly were associated with changes in the free sulphydryl group content of the assembled crude microtubule protein; increased assembly was associated with a higher free sulphydryl group content, decreased assembly with a lower content. 4. 65Zn was found to bind to tubulin. This binding was partly inhibited by N-ethylmaleimide.
Explore the source record for details and available documents.
Immunofluorescence studies of bovine chromaffin cells in culture with specific antibodies against dopamine-beta-hydroxylase gave a distinct punctate pattern of labelling, reflecting the distribution of chromaffin granules. There was strong staining of cell extensions and growth ones. Linear arrays of fluorescent dots were observed, suggesting an association of granules with a filamentous cytoskeleton. Labelling of neuritic processes was periodic, perhaps indicative of a packaging of secretory granules. Chromaffin cells stained strongly with specific anti-actin antisera. Fine filament bundles were observed, and also diffuse staining, some punctate labelling and staining of the plasma membrane or sub-membranous cytoplasm. Growth cones and non-terminal cytoplasmic varicosities contained significant amounts of actin. Colchicine (5 X 10(-5) M) caused retraction of neuritic extensions and formation of lateral growth cones. Cytochalasin (10 microgram/ml) caused ballooning of terminal growth cones and non-terminal cytoplasmic varicosities. Phalloidin (10(-4) M) stimulated microspike formation. The results are discussed in terms of the role of the cytoskeleton in growth cone formation, cell-substratum contacts and the transport of chromaffin granules.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Explore the source record for details and available documents.
Bovine adrenal chromaffin cells were maintained in culture in Dulbecco's modified Eagle's medium containing 20% foetal calf serum and 10 units per ml of Nerve Growth Factor. Under these conditions, chromaffin cells developed up to five neurites per cell. The neurites showed lateral branches and varicosities along their trunk which ended with thick growth cone-like structures. Cultures of chromaffin cells were stained by indirect immunofluorescence with antibodies against (a) chromogranin A to follow the distribution of chromaffin granules, the catecholamine-storing organelles, and (b) tubulin, to study the microtubular system during outgrowth of neurites. Chromogranin A antibodies showed a very intensely staining punctate pattern, not randomly distributed but localized in neurites. Chromaffin granules were found to migrate from the cell body to reach neurite endings where they were densely packed. Intense staining was also observed in varicosities; a linear arrangement of granules was evident along neurite trunks. Tubulin antibodies decorated a complex network, clearly visible at the cell periphery and also in the growth cone-like structures, in the palm region of the growth cone. Colchicine treatment effected retraction of neurites and disappearance of organized microtubule networks; chromaffin granules were found in the perinuclear region of the cell. Some tubulin (0.2% of total membrane proteins) was found in the purified chromaffin granule membrane preparation; however, this tubulin is probably associated with contaminating plasma membranes. By the criteria of morphology and staining with antitubulin antibodies, adult bovine chromaffin cells in culture display characteristics similar to those of sympathetic neurones. In addition, they showed an exaggerated transport of granules. Adult bovine chromaffin cells in culture offer an excellent model for studying the role of microtubules and the contractile apparatus in relation to cell morphological changes and neurosecretion.
Dopamine-beta-hydroxylase, chromogranin A and myosin were purified from bovine adrenal medulla and antibodies prepared against these proteins. Indirect immunocytochemical methods were used to localize dopamine-beta-hydroxylase, chromogranin A and myosin in bovine adrenal medulla and myosin in rat adrenal glands and cells from rat C.N.S. maintained in primary culture. Dopamine-beta-hydroxylase and chromogranin A were found in chromaffin granules, in agreement with biochemical data and, using electron microscopy, dopamine-beta-hydroxylase was found within the matrix and in the surrounding membrane of the storage granule, whereas chromogranin A was confined to the granule matrix. Myosin was localized in the vascular system irrigating adrenal glands, fibroblasts lining the vessels and chromaffin cells. In chromaffin cells, staining was found at the cell boundaries and electron microscopy showed myosin to be associated with the plasma membrane. Faint immunocytochemical staining by antimyosin antibodies was observed around certain exocytotic profiles but particular association with such structures was not demonstrable. Myosin localization was also studied in bovine adrenal cortex, where it was found in vascular channels and faintly in adrenal cortical cells, as in rat adrenal cortex and medulla, where identical patterns were obtained. In neuronal and glial cells dissociated from 13 day rat embryo cerebral hemispheres and cultured for 48 h, localization of myosin was studied using immunohistochemistry. The neuritic expansions and growth cones of neurons were fluorescent, whereas in glial cells, filamentous networks were visualized enclosing the nucleus and as long fibres traversing the entire cytoplasm.
Explore the source record for details and available documents.
Exocytosis was studied in acetylcholine-stimulated bovine adrenal medulla. During a pre-exocytotic stage, chromaffin granules are found in juxtaposition to the plasma membrane and separated from it by an electron dense space 25--27 A in width. Freeze-fracture studies show this stage to be characterized by connections between the granules and the plasma membrane. These connections are apparently cytoplasmic but bridge both membranes; they are presumably proteinaceous, but their exact nature remains to be elucidated. Later stages of exocytosis were also studied by the freeze-fracture technique; a typical feature is the lack of intramembrane particles around the fusion site. Both connections and membrane particle movement are discussed in terms of recent biochemical findings.
Adrenal medullary and retinal myosins formed bipolar filaments in vitro. These filaments showed features suggesting flexibility in the rod region of the myosin molecules composing such filaments; in certain cases the myosin heads spread away from the filament backbone, in others the backbone itself was twisted. In addition the bare central backbone showed transverse striations.
Explore the source record for details and available documents.
Myosin has been isolated from bovine retinae and characterised by its ATPase (ATP phosphohydrolase, EC 3.6.1.3) activity, its mobility in sodium dodecyl sulphate polyacrylamide gels and by electron microscopy. The purified myosin shows high ATPase activity in the presence of EDTA or Ca2+ and a low activity in the presence of Mg2+. The Mg2+-dependent ATPase activity is stimulated by rabbit skeletal muscle actin. The presumptive retinal myosin possesses a major component which has a mobility in sodium dodecyl sulphate polyacrylamide gel electrophoresis similar to that of the heavy chain of bovine skeletal muscle myosin. Electron microscopy showed retinal myosin to form bipolar filaments in 0.1 M KCl. It is concluded that the retina possesses a protein with enzymic and structural properties similar to those of muscle myosin.
Striatal dopamine and its metabolites were studied in rats given lithium chloride in the diet. Results showed an increase in homovanillic acid and 3,4-dihydroxyphenylacetic acid levels but no significant change in dopamine concentration after 3 weeks of lithium administration. There was no change in tyrosine hydroxylase activity after 1, 2, and 3 weeks treatment. The results indicate an increase in the release and turnover of dopamine in the lithium-treated animals.