Chloramphenicol has been used to study mitochondrial biogenesis in mammalian cells by examining its effect on: the incorporation of radioactive amino acids into protein by isolated mitochondria, the growth of L cells, the level of representative enzymes and cytochromes in the mitochondria and cytoplasm and the structure of mitochondria and L cells. A reversible inhibition of synthesis of cytochrome c oxidase was obtained by treating cells with D-threo-chloramphenicol for 90 hr. Recovery of cytochrome c oxidase activity was inhibited by cycloheximide, an inhibitor of cytoplasmic protein synthesis. Cycloheximide also reversibly inhibited cytochrome c oxidase formation in cells which were not treated with D-chloramphenicol. It is suggested that the mitochondria and the nucleus have a joint control in the formation of a functionally active cytochrome c oxidase enzyme. / Thesis / Doctor of Philosophy (PhD)

<p> Monomeric ribosomes and very few subunits are found in mammalian cells and because subunits are required for initiation of protein biosynthesis in both mammalian and bacterial systems, this implies that the dissociation step in the ribosome cycle does not occur spontaneously. Our attention was drawn to the possibility that the monomeric ribosomes in mammalian cells could complex with a dissociation factor. This factor would perhaps be present in the cell in limited supply and would, therefore have to recycle in the course of initiation, from a completed initiation complex to another free ribosome. An assay was set up whereby the existence of a dissociation factor in a subcellular fraction of rat liver could be determined. The perfecting of the assay system for the dissociation factor yielded much information on the ionic concentration necessary for both ribosome and subunit stability. The factor was found to be present in the fraction containing the "native" subunits. This is identical to the situation which exists in E. coli. The factor is capable of dissociating rat liver monomeric ribosomes into 60S and 40S subunits. The factor was found to act on ribosomes freed of both messenger RNA and nascent protein.</p> <p> Purification of the crude dissociation factor preparation was achieved by obtaining at 4°C the 35-65% ammonium sulphate fraction. Purification was also achieved by means of an incubation of the preparation at 40°C for 30 minutes followed by a centrifugation to remove precipitated protein.</p> <p> The DF was determined to have a molecular weight in excess of 85,000 by column chromatography.</p> / Thesis / Master of Science (MSc)

Mammalian herbivory of bottomland hardwood seedlings has been listed as one of the primary causal factors of failed afforestation efforts in the Lower Mississippi Alluvial Valley (LMAV). This study examined the biological and economic effects of mammalian herbivory on recently afforested carbon sequestration sites in the LMAV. Selected seedlings of six planting mixes were observed through the first year following planting to monitor seedling survival, growth, and mammalian herbivory. It was determined that greater than 10% of selected seedlings were browsed by various mammalian herbivore species, and some species mixes were browsed in excess of 50%. Financial analyses compared alternative afforestation strategies and determined to what extent herbivore-induced seedling mortality could reduce investment returns of landowners engaged in afforestation activities. In the presence of extreme mammalian herbivory, landowner returns can be reduced by hundreds of dollars per acre and could prevent further afforestation activities in the LMAV.

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No description available.

<p> An area of current research interest involves the diacylglycerol kinase (DGK) family. Diacylglycerol kinases (DGKs) are a group of enzymes that phosphorylate diacylglycerol (DAG), a second messenger involved in cell signaling. The product of this reaction, phosphatidic acid (PA), also has signaling roles. An interesting isoform is DGKε, that although it has no identifiable regulatory domains other than the C1 domains. In addition, the catalytic domain is homologous to that of other DGK isoforms; however, DGKε exhibits an unusual specificity toward acyl chains of DAG, selectively phosphorylating an arachidonoyl-DAG substituted at the sn-2 position. Recently, researchers have identified an N-terminal hydrophobic domain of about 19 amino-acids in human DGKε. The present study attempted to identify the function of the N-terminal putative transmembrane domain of human DGKε and its relationship to the activity and substrate specificity of this enzyme by designing a truncated form of DGKε lacking the putative transmembrane domain.</p> <p> We have shown that the putative transmembrane domain of DGKε is not required for enzyme activity or for substrate specificity. In a mixed micellar assay the enzyme-catalyzed reaction followed surface dilution kinetics with respect to diacylglycerol and followed Michaelis-Menten kinetics with respect to ATP. The results show that the truncated form of the enzyme maintains substrate specificity for lipids with an arachidonoyl moiety present at the sn-2 position. The truncation increased the catalytic rate constant for all three substrates used in this study. It appears unlikely that the putative transmembrane domain, a segment unique to DGKε, has no functional role. It is possible that the hydrophobic segment may have a role in enzyme regulation by associating the enzyme in oligomers that are inactive in quiescent cells and get activated upon dissociation into monomers by increased levels of DAG in the membrane. We have shown that the presence of higher molecular species in the gel is not dependent on the presence or absence of the putative transmembrane domain. The only difference between the full-length and truncated enzyme is the monomer to dimer ratio. It appears likely that another segment of DGKε besides the putative transmembrane domain may be involved in oligomerization and that oligomerization is either transient or very weak. The absence of the hydrophobic domain of DGKε seems to cause no drastic changes either in the activity, the substrate specificity, or the state of oligomerization of the enzyme.</p> <p> Therefore, the next question is whether the hydrophobic domain of DGKε inserts itself in the membrane as a transmembrane helix or it only helps associate the enzyme to the surface of the membrane. We studied the topology of theN-terminal domain of DGKε in intact and permeabilized cells by indirect immunofluorescent microscopy. The results show that the N-terminal domain of the protein is present in the cytosol. The data supports a model in which the hydrophobic domain of DGKε forms a hydrophobic loop that attaches to the inner layer of the plasma membrane or that the hydrophobic domain attaches to the inner leaflet through its nonpolar surface of a horizontal helix. The first hypothesis is supported by the presence of a Pro residue in the middle of the hydrophobic domain. This Pro would introduce a kink in the helix creating a loop, but the absence of one or more glycine residues proximal to proline may hinder the formation of the loop. The second hypothesis is sustained by the presence of a polar surface on one side of the helical wheel. This orientation indicates the presence of a slightly horizontal helix attached to the surface of the inner layer of the plasma membrane.</p> <p> Regardless of the orientation of the helix, the weak association of the enzyme with the membrane is supported by previous data on the ease of extractability of the enzyme with high salts and on the Triton X-114 phase partitioning.</p> / Thesis / Master of Science (MSc)

Bcl-XL is a membrane-associated protein that inhibits programmed cell death (apoptosis) in mammalian cells. Very little is known about the membrane topology of Bel-XL or how its association with membranes contributes to its function. It was the aim of this thesis to use fluorescence spectroscopy to investigate the location of a specific amino acid ofBcl-XL relative to the membrane. Bel-XL purified from E. coli could bind both to large unilamellar vesicles and endoplasmic reticulum (ER) microsomes isolated from canine pancreas. A cysteine residue at position 151 in Bcl-XL could be covalently labelled with the environmentally sensitive fluorescent molecule NBD. Emission intensity measurements in the presence and absence of membranes, combined with aqueous and lipophilic quenching experiments, indicate that Cys 151 is inserted into the interior of the membrane bilayer when Bcl-XL is bound to membranes. The methods outlined in this thesis form the basis for an experimental system that can be used to determine the membrane topology ofBclXL under a variety of conditions. / Thesis / Master of Science (MSc)

Cell proliferation and differentiation are tightly regulated processes required for the proper development of multi-cellular organisms. To understand the effects of cell proliferation on embryo patterning in mice, we inactivated Aurora A, a gene essential for completion of the cell cycle. We discovered that inhibiting cell proliferation leads to different outcomes depending on the tissue affected. If the epiblast, the embryonic component, is compromised, it leads to gastrulation failure. However, when Aurora A is inactivated in extra-embryonic tissues, mutant embryos fail to properly establish the anteroposterior axis. Ablation of Aurora A in the epiblast eventually leads to abnormal embryos composed solely of extra-embryonic tissues. We took advantage of this phenomenon to generate embryonic stem (ES) cell-derived mice. We successfully generated newborn pups using this epiblast ablation chimera strategy. Our results highlight the importance of coordinated cell proliferation events in embryo patterning. In addition, epiblast ablation chimeras provide a novel in vivo assay for pluripotency that is simpler and more amenable to use by stem cell researchers.

Photocontrol, the use of light-sensitive proteins to control events within living tissue, allows complex processes in higher organisms to be studied. The Halorhodospira halophila photoactive yellow protein (PYP) can be used to regulate transcription factor activity with blue light. Before any PYP-based system can probe complex processes in higher organisms, proof of functional expression in vivo is required. We linked d25 PYP to the C-terminus of blue fluorescent protein (BFP) and expressed variants of the fusion protein (BFPd25PYP) in E. coli and human embryonic kidney (HEK293) cells. Expression of BFPd25PYP in E. coli verified in vitro photoswitching. The fusion protein was successfully expressed in HEK293. Fluorescence studies of intact cells indicated chromophore uptake and incorporation into PYP in HEK293, while photoswitching of PYP was measured in protein isolated from HEK293. These findings are promising for the development of applications using PYP for in vivo mammalian photocontrol of biological events.

Photocontrol, the use of light-sensitive proteins to control events within living tissue, allows complex processes in higher organisms to be studied. The Halorhodospira halophila photoactive yellow protein (PYP) can be used to regulate transcription factor activity with blue light. Before any PYP-based system can probe complex processes in higher organisms, proof of functional expression in vivo is required. We linked d25 PYP to the C-terminus of blue fluorescent protein (BFP) and expressed variants of the fusion protein (BFPd25PYP) in E. coli and human embryonic kidney (HEK293) cells. Expression of BFPd25PYP in E. coli verified in vitro photoswitching. The fusion protein was successfully expressed in HEK293. Fluorescence studies of intact cells indicated chromophore uptake and incorporation into PYP in HEK293, while photoswitching of PYP was measured in protein isolated from HEK293. These findings are promising for the development of applications using PYP for in vivo mammalian photocontrol of biological events.