O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and ubiquitous post-translational modification that has been shown to play a role in regulating a variety of intracellular processes. The pathway responsible for generating the O-GlcNAc modification, the hexosamine biosynthetic pathway (HBP), has also been shown to affect the activity and translocation of certain protein kinase C (PKC) isoforms. To investigate if the effects of HBP flux on PKC translocation observed by others is related to the O-GlcNAc modification, O-GlcNAc levels in human astroglial cells were elevated using four separate O-GlcNAc modulating agents followed by analysis of cytosol and membrane concentrations of PKC-epsilon, -alpha, -betaII, and -iota. Of the four PKC isoforms analyzed, PKC-epsilon showed a significant reduction in its membrane associated levels in response to all agents tested whereas PKC-alpha showed reductions in response to only two agents. Investigation of the mechanism for the reductions in membrane associated PKC-epsilon and -alpha indicate that the increased O-GlcNAc levels did not disrupt the activation of these isoforms or their ability to translocate to the plasma membrane. Furthermore, results indicate that these reductions are not due to a disruption in the Hsp70 mediated recycling of the isoforms. It was found; however, that increased O-GlcNAc levels resulted in increased degradation of PKC-epsilon suggesting that the decreases in membrane associated PKC-epsilon may be a result of increased phosphatase or protease activity. Additional studies revealed that decreases in membrane bound PKC-epsilon and PKC-alpha, both of which act as anti-apoptotic enzymes, correlated with an increase in poly-(ADP-ribose) polymerase (PARP) cleavage -- a well characterized hallmark of apoptosis. In addition to PKC, the effects of increased O-GlcNAc levels on a related kinase, Akt, were also examined. Initial investigation of the effects of increased O-GlcNAc modification of Akt activation using glucosamine or streptozotocin revealed a relatively large, short-term increase in Akt phosphorylation in response to these treatments. However, further analysis with other O-GlcNAc modulators indicated that this activation was not related to O-GlcNAc protein modification. Furthermore, this activation does not appear to be related to any hyperosmotic effects associated with the treatment conditions, nor does it appear to be related to oxidative stress. Therefore, further investigation is needed to characterize the novel pathway responsible for Akt activation following glucosamine or streptozotocin treatment.

The biosynthesis of proteins occurs at the ribosomes, where amino acids are linked together into linear chains. Nascent protein chains may undergo several different processes during their synthesis. Some proteins begin to fold, while others interact with chaperones, targeting factors or processing enzymes. Nascent membrane proteins are targeted to the cell membrane for integration, which involves the translocation of periplasmic domains and the insertion of membrane-embedded parts. The aim of this thesis was to gain insights about the dynamics of nascent peptide chains undergoing folding, membrane translocation and integration. To this end, we explored the use of arrest peptides (APs) as force sensors. APs stall ribosomes when translated unless there is tension in the nascent peptide chain: the higher the tension, the more full-length protein can be detected. By using APs, we could show that a transmembrane helix is strongly ‘pulled’ twice on its way into the membrane and that strong electric forces act on negatively charged peptide segments translocating through the membrane. Furthermore, we discovered that APs could be used to detect protein folding and made the surprising discovery that a small protein domain folded well inside the ribosomal tunnel. Finally, we explored the arrest-stability of a large set of AP variants and found two extremely stable APs.

During the synthesis of secretory and membrane proteins, polypeptides move through a universally conserved protein-conducting channel, formed by the Sec61/SecY complex that is located in the eukaryotic endoplasmic reticulum membrane or the prokaryotic plasma membrane. The channel operates in two different modes depending on its binding partners. In co-translational translocation, a pathway found in all organisms, the channel associates with a translating ribosome. In post-translational translocation, the channel cooperates with either the Sec62–Sec63 complex in eukaryotes or the SecA ATPase in bacteria. Despite tremendous progress in our understanding of protein translocation over the past decades, many questions about its mechanism remain to be answered. These include (1) how the channel maintains the membrane barrier for small molecules while transporting large proteins, (2) what is the functional implication of channel oligomerization, and (3) how the channel interacts with binding partners and polypeptide substrates during translocation. To address these questions, we developed a novel in vivo method to generate both co- and post-translation translocation intermediates in intact Escherichia coli cells, such that polypeptide chains are only partially translocated through the channel. Using this method, we first demonstrated that a translocating polypeptide itself blocks small molecules from passing through an open SecY channel. A hydrophobic pore ring surrounding the polypeptide chain is vital for maintaining the membrane barrier during translocation. Next, we examined the importance of SecY oligomerization in protein translocation. Crosslinking experiments showed that SecY molecules interact with each other in native membranes, but that this self-association is greatly decreased upon insertion of polypeptide substrates. We also showed that SecY mutants that cannot form oligomers are still functional in vivo. Collectively, our data indicate that a single copy of SecY is sufficient for protein translocation. Finally, we isolated an intact co-translational translocation intermediate from E. coli cells and analyzed its structure by cryo-electron microscopy. An initial map shows a translating ribosome containing all three tRNAs is bound to one copy of the SecY channel. Analysis of a large dataset is ongoing in order to understand the structural basis of how the channel interacts with the ribosome and translocating nascent chain.

A DNA molecule passing through a nanopore in a liner and sequential fashion allows for unprecedented interrogation of the polymer. Adding transverse electrodes that are comparable in size and sensitive to the DNA molecule, can further the attempts to rapidly sequence DNA. Carbon nanotubes are comparable in size and interact strongly with the DNA molecule. This makes them an excellent choice for integration with nanopores. Only the section of the carbon nanotube in immediate proximity to the nanopore should be sensitive to the DNA molecules. Atomic layer deposition of metal-oxides passivates the sections of the carbon nanotube that are not to interact with the DNA molecule. The coating also protects the thin film interconnects leading to the carbon nanotube. Hafnium oxide is superior to aluminum oxide in chemical resistance and electrical insulation but leads to a high failure rate of the carbon nanotube across nanopore devices. Aluminum oxide, combined with gold thin film interconnects to the carbon nanotube, produced the first functioning devices in electrolyte. These devices had concurrently functioning ionic (current across the nanopore) and transverse (current through the carbon nanotube) channels. No concurrent DNA translocation signal was recorded on the ionic and nanotube current traces. Analyzing the translocation events recorded on the ionic channel indicated that double-stranded DNA (dsDNA) passed through the carbon nanotube articulated nanopore an order of magnitude slower than it would have through a comparable unarticulated nanopore. The slower translocation observed is a necessary condition for sequencing. Investigating dsDNA translocation under various experimental conditions led to the discovery of a new interaction between the molecule and small nanopores. A dsDNA molecule is trapped when the electric field near the nanopore attracts and immobilizes a non-end segment of the molecule at the nanopore orifice without inducing folded translocation. In this work, the expression “trapped dsDNA” will exclusively refer to the immobilization of a dsDNA molecule at the orifice of the nanopore. The ionic current through the nanopore decreases when the dsDNA molecule is trapped by the nanopore. By contrast, a translocating dsDNA molecule under the same conditions causes an ionic current increase. Finite element modeling results predict this behavior for the conditions of the experiment. / Physics

No description available.

A review of the literature established that there were areas of cytokinin transport which needed further investigation, in order to determine the function and/or mode of action of cytokinins during certain stages of plant development. Radioactive zeatin was applied to plant systems suitable for determining more about specific problems of cytokinin transport. The metabolism of the radioactive zeatin was monitored in relation to transport. The metabolism in, and possible export of, radioactive zeatin out of immature, mature and senescing Ginkgo biloba leaves was monitored using explants. The results showed that approximately the same percentage radioactivity was exported from the leaves at all three stages of their development. This indicates that these deciduous leaves could potentially export cytokinins, but the results were not regarded as being significant. Cytokinins would not have been expected to be transported out of expanding leaves, which rapidly utilize cytokinins, and in comparison greater cytokinin export would have been expected to occur from senescing leaves, but this did not occur. The resuIts could indicate that cytokinin glucosides in deciduous leaves are primarily inactivation products rather than storage compounds. The metabolism and transport of radioactive zeatin, applied to the leaves of Citrus sinesis trees, was monitored during the flush of new growth following a dormant period. Some of the radioactive zeatin applied to these leaves appeared to be utilized in the new shoot growth. This could imply that accumulated cytokinin glucosides in these evergreen leaves are exported out of the leaves and reutilized, and thus fulfil a storage function; although the extent to which export occurred as opposed to interconversion and/or catabolic metabolism in the leaves could not be determined. The distribution of radioactive zeatin applied to the xylem and phloem of Phaseolus vulgaris plants at three stages of development was also monitored. There appeared to be preferential transport of cytokinins in the transpiration stream, that is, applied to the xylem. The vegetative apices, buds and flowers appeared to be the major sinks for radioactive compounds in the vegetative and flowering plants, and the leaves were the most important sink in the fruiting plants. Radioactive zeatin in the phloem appeared to move passively with the assimilate stream. The transport of radioactive zeatin applied to the primary leaves appeared to confirm that cytokinins in the phloem were transported along with the assimilate stream. The results also indicated that the accumulated cytokinin glucosides in these annual leaves were primarily inactivation products, involved in regulating leaf metabolism, although some re-utilization of these cytokinins could potentially take place. could have reflected normal cytokinin transport occurring in the tissues. The main metabolic pathway, oxidation, did not, however, appear to be the main metabolic pathway in the plant tissues and may have been the result of the exogenous application of zeatin. The fact that metabolism did not appear to be the normal metabolism of the tissues, could potentially have affected transport. / Thesis (Ph.D.)-University of Natal, Pietermaritzburg, 1982.

No description available.

Saflufenacil is a new herbicide for broadleaf weed control. Limited information is available for crop tolerance, weed control and herbicide behavior in the rice environment. Studies were designed to 1 and 2) evaluate rice tolerance and weed control to saflufenacil in combination with clomazone and imazethapyr; 3) evaluate the absorption and translocation of imazethapyr and saflufenacil in weed species 4) assess saflufenacil degradation and persistence in soils; and 5) investigate the use of reference compounds during the determination of pesticide adsorption (Kd). None or minimal rice injury was observed from preemergence (PRE) application of saflufenacil. Intense injury (68%) was noted with combinations of clomazone (505 g ha-1) applied PRE and saflufenacil (50 g ha-1) applied postemergence (POST). Similarly, rice injury up to 83% was observed in earlier evaluations when saflufenacil was applied POST with imazethapyr. However, subsequent evaluations indicated rice recovery from herbicide treatments. Combination of saflufenacil with imazethapyr resulted in hemp sesbania control ≥ 88% and red rice control of 100%. Rice yield was not adversely altered by the herbicide treatments used in the clomazone and imazethapyr weed control programs. Imazethapyr plus saflufenacil provided a greater uptake (30%) and translocation (35%) of 14C-imazethapyr than imazethapyr alone in the TX4 red rice. Absorption of 14C-saflufenacil ranged from approximately 40 to 60% in hemp sesbania plants. At 12 and 24 hours after treatment a greater percentage of the absorbed saflufenacil was quantified above the treated leaf at the two lower light intensities. Similar trends were observed for basipetal movement of saflufenacil. An accelerated solvent extraction method was developed to extract saflufenacil from soil. Half-life averaged among soils was 59 and 33 days for saturated and field capacity, respectively. Saflufenacil persistence in the environment was 2 to 3 times longer under flooded conditions for most of the studied soils. Adsorption values were affected by soil to solution ratios, particularly when the soil-pesticide interaction resulted in Kd values > than 2 mL g-1. The use of reference compounds during Kd estimation allowed for calculation of a conceptual adsorption window generating a more comprehensive set of data with alternatives for comparison of soils and methods.

Deletion of the long arm of chromosome 6 is one of the most common genetic alterations in human malignant melanoma. Recently, a reciprocal translocation between chromosomes 6q and 17p was detected in a melanoma cell line, UACC-930, using arm painting probes of 6p and 6q. Reciprocal translocation is seldom observed in solid tumors. Upon further characterization of the translocation marker using techniques such as Southern blotting, genomic library screening and DNA sequencing, a complex rearrangement including two inversions of 6q and a translocation between the inverted 6q and 17p, [der(6)inv(6)(q21q22)(q22q27)t(6;17)(q27;p13)], was detected. An NCBI blast search revealed 3 genes being interrupted by the breakpoints: prenyl diphosphate synthase subunit 2 (PDSS2) at 6q21, Parkin at 6q27 and p53 at 17p13. Down-regulation of PDSS2 was commonly observed in 59/87 (67.8%) primary melanomas, which was significantly higher than that in benign nevi (7/66, 10.6%, p<0.001), indicating the tumor-suppressive potential of PDSS2 in melanoma development. To characterize the function of PDSS2 in tumorigenesis, PDSS2 was stably transfected into a highly tumorigenic melanoma cell line, UACC-903. The tumor-suppressive function of PDSS2 was demonstrated by both in vitro and in vivo assays. The results showed that PDSS2 could inhibit tumor cell growth, decrease the colony-forming ability in soft agar, and totally abrogate the tumorigenicity of UACC-903 in nude mice. PDSS2 is the first enzyme involved in the CoQ10 biosynthesis pathway. Other studies have demonstrated PDSS2 mutations can cause severe CoQ10 deficiency and markedly reduced ATP production because of respiratory chain dysfunction. Interestingly, proteomics analysis revealed 7 out of 11 identified proteins (HSPA8, GAPDHS, TPI1, HSPA5, PGK1, ENO1, and ATP5B) differentially expressed in PDSS2-overexpressing cells were related to energy metabolism. Further studies are required to determine how PDSS2 could alter the energy supply in tumor cells. Taken together, these results support the proposal that PDSS2 is a novel tumor suppressor gene which may play an important role in the development of malignant melanoma via altering tumor metabolism.

This thesis presents the results of an investigation into the uptake and distribution of cadmium (Cd) in two cultivars of potato (Solanum tuberosum L.) shown to contain different concentrations of Cd in the tuber at maturity. An initial glasshouse trial sought to determine whether differences in tuber Cd between these two cultivars resulted from differences in uptake from the soil, or were due to differences in the allocation of Cd to the various tissues within the plant. Total uptake of Cd from the soil did not differ between cultivars, nor did the yield of tubers. However, there were marked differences in Cd distribution within the plant. Most of the differences in tuber Cd concentration could be accounted for by a large (3-fold) retention of Cd in the roots of cultivar Wilwash. The concentration of Cd in the shoots of Wilwash was also higher than of Kennebec, although to a lesser extent than the roots. Further studies were conducted to trace the pathways of Cd uptake and movement within the plant. A split-pot trial, involving long-term growth of potatoes in 109Cd-labelled soil, was undertaken to determine the overall pattern of Cd distribution and the importance of the root system in supplying Cd to the tubers. The root system of the potato plant is different to many plants, in that the main root system (basal roots) is augmented after tuber initiation by roots extending from the stolon and from the tuber itself. The basal roots were found to be the dominant source of Cd to all tissues and accounted for approximately 85 % of tuber Cd. The remaining tuber Cd was sourced directly from the stolon and tuber roots. However, there was no evidence of a direct link between the main (basal) root system and the stolons. Although Cd was found to accumulate in the periderm of the tubers, there was no uptake into the tuber tissue itself. Isotopic studies were undertaken to investigate the short-term movement of newly absorbed Cd in the xylem and the phloem. Cadmium was found to be highly mobile in both the xylem and phloem, with added Cd being rapidly assimilated into all tissues following both root and foliar application. Newly absorbed Cd was rapidly sequestered by the stems when applied to either the soil or to a source leaf, suggesting that the stems may act as a transitional storage pool when rapid turnover of nutrients and other mineral elements is required during tuber bulking. Inhibition of Cd uptake by zinc (Zn), has been proposed as a method for reducing the concentration of Cd in various agricultural crops, including potatoes. The ability of Zn to reduce Cd uptake was found to be highly dependent upon cultivar and on the concentration of Cd in the external medium. Although competition between Zn and Cd was found for cultivar Wilwash when the external concentration of Cd was low, when the concentration of Cd in the external media was high, increasing Zn served to increase Cd uptake. Both synergistic and competitive responses were also noted for cultivar Kennebec. However, the patterns of response were opposite to those evident in Wilwash. The complexity of these interactions highlighted the possible shortcomings in using soil applied Zn to limit Cd uptake by potatoes. / Thesis (Ph.D.) -- University of Adelaide, School of Earth and Environmental Sciences, 2004.