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Biomedical subjects

R W Parish

Publications and source records attributed to R W Parish.

At least 55 records · Page 3Linked to original sources
PubMed1979

Cyclic AMP induces the synthesis of developmentally regulated plasma membrane proteins in Dictyostelium.

Dictyostelium discoideum slugs (pseudoplasmodia) were disaggregated and the resynthesis of developmentally regulated plasma membrane proteins examined. The synthesis of the majority of these proteins was inhibited when cells were overlaid with Cellophane and maintained as a monolayer. However, cell contact and movement did occur under the Cellophane. The inhibition of differentiation may result from the inability of the cells to organize specifically into multicellular aggregates. The addition of cyclic AMP (1--5 mM) induced the synthesis of certain developmentally regulated plasma membrane proteins in cells overlaid with Cellophane. Hence, this confirms other work showing that cyclic AMP is required for at least some post-aggregative gene expression. Specific cell organisation and interactions are apparently required for an increase in or maintenance of intracellular cyclic AMP levels.

Cell Membrane↗
PubMed1977

Inhibition of Dictyostelium discoideum beta-glucosidase by purines.

beta-Glucosidase of Dictyostelium discoideum is inhibited by purines in the following order: adenine greater than adenosine greater than 6-methylaminopurine greater than hypoxanthine greater than inosine greater than purine greater than guanosine. Adenine inhibits activity by 50% at 1 to 2 mM. The kinetics are complex because the enzyme is stimulated by substrate and inhibited by glucose.

Adenine↗
PubMed1976

Hyphal tip growth in Phytophthora. Gradient distribution and ultrahistochemistry of enzymes.

Germinating cysts and isolated walls from germinating cysts incorporated 14C-UDPG into wall material of which 22.5 and 15% respectively were insoluble in boiling 1 N HCl, indicating that part of the synthetase activity is located in the wall itself. A combination of Urografin and Ficoll density gradients was used to separate various intracellular fractions. A consistent separation of beta-glucanase and UDPG-transferase enriched fractions was achieved. The beta-glucanase fraction contained dictyosome vesicles and fragments along with some plasma membranes. The UDPG-transferase fraction was relatively rich in membranes resembling rough and smooth ER. The results suggest the two enzymes are transported to the wall by different intracellular routes, and two types of vesicle may be involved. Alkaline phosphatase, beta-glucosidase and acid phosphatase were found extracellularly and their distribution in density gradients determined. The results of histochemical staining for acid phosphatase, alkaline phosphatase and polysaccharide are described and compared with the biochemical data. beta-1,3-glucanase, found intra- and extracellularly, induced distorted growth of germ tubes and also removed most of the apical wall when added to the incubation medium. None of these responses were observed with cellulase. Determinations of the osmotic pressure of germinating cysts and incubation medium revealed that the turgor of germinating cysts amounts to about 1.8 at under the conditions used.

Acid Phosphatase↗
PubMed1976

A labile acid phosphatase isozyme associated with the surface of vegetative Dictyostelium discoideum cells.

A minor acid phosphatase isozyme (acid phosphatase I) of vegetative Dictyostelium discoideum amebae has been shown to be associated exclusively with the external surface of the plasma membrane. The isozyme is not present in phagocytic vacuoles isolated with latex beads. The isozyme disappears from cells removed from nutrient medium and does not reappear during differentiation. When inhibitors of protein synthesis (e.g. cycloheximide, chloral hydrate, concanavalin A) are added to cells growing in nutrient medium, acid phosphatase I is rapidly lost. It appears that the level of protein synthesis need only be moderately reduced (less than 25%) to induce loss of enzyme activity. Treatment with inhibitors of DNA and RNA synthesis for up to 2 h had no effect on isozyme activity. It is postulated that the cells are able to "sense" (through the reduction in levels of protein synthesis) when external conditions become unfavorable, and immediately respond by reducing the activity of enzymes involved in maintaining contact with the extracellular environment. The closed system thought necessary for differentiation would then be created.

Acid Phosphatase↗
PubMed1975

The isolation and characterization of peroxisomes (microbodies) from baker's yeast, Saccharomyces cerevisiae.

Peroxisomes were isolated form derepressed (lactose grown) Saccharomyces cerevisiae cells following homogenization with a "Merkenschlager" cell mill (at 0 degrees C using glass beads). Catalase and urate oxidase, along with low activities of D-amino acid oxidase and L-alpha-hydroxyacid oxidase (glycollate oxidase), were associated with the peroxisomes. No catalase activity was present in glucose repressed cells. When protoplasts prepared from derepressed cells were used for peroxisome isolation, catalase activity was not sedimentable through gradients. Apparently peroxisomes were destroyed as the cells became fermentative during protoplast preparation. The distribution of glyoxylate cycle enzymes was examined. Isocitrate lyase was not sedimentable, suggesting that, if the enzyme is peroxisome-associated, it is either readily released of present in a labile second class of peroxisomes. Low activities of malate dehydrogenase and citrate synthetase were found in peroxisome fractions from gradients, but may represent mitochondrial contamination. Citrate synthetase was not found associated with a low-density particle as had been previously reported.

Alcohol Oxidoreductases↗
PubMed1975

Mitochondria and peroxisomes from the cellular slime mould Dictyostelium discoideum. Isolation techniques and urate oxidase association with peroxisomes.

The isolation of cell organelles from Dictyostelium discoideum was attempted using a variety of techniques. Cell homogenization (e.g. Potter-Elvehjem, glass beads) gave poor yields of organelles which were, in addition, exceptionally fragile and unstable in density gradients. An isolation method was developed using Triton X-100 in buffered sorbitol/Ficoll solutions at concentrations optimal for plasma membrane rupture. Immediately following cell lysis the solutions were diluted to sub-optimal Triton X-100 concentrations. Sedimentabilities of malate dehydrogenase, citrate synthetase, urate oxidase and catalase of around 55%, 40%, 35% and 55% respectively could be demonstrated using this method. The organelles were more resistant to breakage during resuspension following differential centrifugation and remained largely intact during density gradient centrifugation. The distribution of adenylate kinase activity in gradients showed that at least half the mitochondria retained an intact outer membrane. The mitochondria and peroxisomes could not be clearly separated using conventional sucrose-Ficoll density gradients. Separation was achieved by incubating the cell homogenate with succinate and a tetrazolium dye (2-p-iodophenyl-3-p-nitrophenyl-5-phenyl monotetrazolium chloride). Succinate dehydrogenase activity of mitochondria reduced the tetrazolium dye and the product (formazan) was deposited on the mitochondrial membranes ("heavy-labelling"). The mitochondria then sedimented to denser regions of the gradient while catalase distribution remained unchanged. The treatment left both organelles intact. The mitochondria (1.21 g/ml) were slightly denser than the peroxisomes (1.19 g/ml). The peroxisomes contained catalase and urate oxidase; no other hydrogen-peroxide-producing oxidases were detected. The slime mould urate oxidase resembled the mammalian enzyme. It had an apparent Km value of 12.5 muM, an optimum of activity at pH 8.5 in borate buffer and was competitively inhibited by trichloropurine.

2,6-Dichloroindophenol↗