Antibacterial agents are composed of a diverse group and many such agents have entered common usage through experience with little information on their mechanism of action. Study of the mechanism of action of an antimicrobial agent provides an insight into resistance mechanisms, toxicological problems and the design and development of new agents or combinations. The primary target of most antimicrobial agents (excluding antibiotics) is the cytoplasmic membrane and associated enzymes. Membrane-active agents can cause a change in the fluidity and/or permeability of the cytoplasmic membrane. Such changes can be determined by the leakage of cellular constituents such as potassium ions, nucleotides and their constituents and amino acids. The effect of an anti bacterial agent on the cytoplasmic membrane can also be determined by elucidating the effect of the antibacterial agent on the activity of membrane-bound enzymes and substrate uptake.

The role of cytoplasmic pH in guard cell signal transduction was investigated in epidermal strips of Commelina communis. The cytoplasmic pH of guard cells was measured by dual excitation ratio confocal laser scanning microscopy. Large transient alkalinisations occurred for up to 20 minutes both during closure, in response to ABA and calcium, and opening in response to IAA and fusicoccin. Therefore the direction of the pH change does not determine the direction stomatal movement in Commelina communis in contrast to previous reports in Paphiopedilum tonsum. Furthermore, CO₂ caused a slow acidification during stomatal closure, indicating that pore movements are not always associated with a transient cytoplasmic alkalinisation. The internal pH of guard cells was buffered by low concentrations of isobutyrate. Small reductions in stomatal closure in response to ABA and calcium were observed, however, responses to CO₂, IAA and fusicoccin were unaltered. High levels of isobutyrate stimulated wide stomatal opening for all stimuli. Therefore manipulation of cytoplasmic pH only give limited support in the case of ABA and calcium that cytoplasmic pH changes are either necessary for or modulate stomatal movements. The observed pH changes may therefore be a consequence of the mechanism underlying pore movement rather than genuine cytoplasmic signals per se, A model is described based on strong ion and weak acid chemistry which predicts that the observed pH transients result from changes in the concentrations of chloride and malate which charge balance the potassium fluxes during stomatal movements. No suitable fluorescent indicator was found to measure pH in either the apoplast or vacuole. However the volume of the guard cell lumen, vacuole, nucleus and chloroplast were directly measured during stomatal movements and the cytoplasmic volume was calculated. These volumes were used to re-calculate compartmental pH and ion concentrations from previous reports.

Many insects and other arthropods have symbiotic microorganisms that may influence key facets of their biology. Cardinium hertigii is an intracellular bacterial symbiont, (phylum Bacteroidetes) of arthropods and nematodes. This versatile symbiont has been shown to cause three of four reproductive manipulations of their arthropod hosts known to be caused by symbionts: parthenogenesis induction (PI), where genetic males are converted into genetic females; feminization, where genetic males become functional females; and cytoplasmic incompatibility (CI), the symbiont-induced death of offspring from matings of infected males and uninfected females. Here, I explored the evolution of this symbiont and its reproductive manipulations, and found that closely related Cardinium strains have a tendency to associate with closely related hosts and the reproductive manipulations do not display a clear phylogenetic signal. To further understand the possible genes underlying these reproductive manipulations, I sequenced four Cardinium genomes and compared these with the two genomes analyzed in the literature. In these comparisons, I found that, although closely related Cardinium strains tend to reside in closely related hosts, there is no evidence for a suite of genes associated with host specificity, as few differences separate two strains residing in different host orders, suggesting that ecological opportunity for horizontal transmission may be more limiting to Cardinium than genomic capability. I additionally identify some genes that may be associated with the Cardinium’s ability to induce PI and CI in its wasp host. Overall, this dissertation has led to a better understanding of Cardinium and its effects on its hosts.

Dissertation (Ph.D.) -- University of Texas Southwestern Medical Center at Dallas, 2008. / Vita. Bibliography: p. 158-174.

The ability of proteins to engage in nuclear-cytoplasmic shuttling is required for their proper function. The nuclear export of the von Hippel Lindau (VHL) tumour suppressor protein is necessary for the proteasomal degradation of the hypoxia inducible factor alpha (HIFα). Studies have identified that the nuclear export of VHL and other proteins encoding a Transcription-Dependent Nuclear Export Motif (TD-NEM) is independent of the classical CRM1 nuclear export pathway but requires ongoing transcription. Furthermore, the eukaryotic elongation factor 1 alpha (eEF1A) was identified as a mandatory component of the TD-NEM-mediated nuclear export machinery. In this study, we have uncovered the ability of eEF1A to mediate the nuclear export of proteins by accessing the nuclear compartment in its inactive, GDP-bound form. Although previously thought of as a strictly cytoplasmic protein, work conducted in this thesis has shown that eEF1A is a nuclear-cytoplasmic shuttling protein and this ability is required for the effective export of proteins encoding a TD-NEM.

Thesis advisor: Laura E. Hake / Cytoplasmic polyadenylation regulates translational activation of mRNA stored in immature Xenopus oocytes. This event is necessary for the beginning of oocyte maturation, and later for critical processes in early embryonic development. A major protein required for polyadenylation is the cytoplasmic polyadenylation element-binding protein (CPEB), which recruits a factor that promotes the interaction between Poly(A) polymerase and the end of the mRNA. Polyadenylation in turn leads to translation through interactions between CPEB and other proteins. Using a yeast two-hybrid screen, several of these proteins were identified and cloned, including two of note. X295, a zinc-finger containing novel protein, and DEK, which has significant homology with the Homo sapiens DEK involved in certain juvenile leukemias. Through the cloning of the genes encoding these proteins, transcription of mRNA, and protein overexpression in oocytes, a series of protein-protein interaction binding assays were performed. Immunoblotting of SDS-PAGE analyzed samples shows that GST-CPEB and HA-X295 interact in ovo, and suggests a possible in ovo interaction of endogenous CPEB and endogenous X295. In similar experiments, DEK and CPEB do not interact, suggesting they may not interact in ovo. / Thesis (BS) — Boston College, 2003. / Submitted to: Boston College. College of Arts and Sciences. / Discipline: Biology. / Discipline: College Honors Program.

Cu+-ATPases receive Cu+ from specific chaperones via ligand exchange and subsequently drive the metal efflux from the cell cytoplasms. Cu+-ATPases have two transmembrane metal binding/transport sites (TM-MBS) and various cytoplasmic domains: the actuator (A-domain) and ATP binding domains (ATPBD), and regulatory N-terminal metal binding domains (N-MBD). Archaeoglobus fulgidus CopA, the Cu+-ATPase used in these studies, contains a single N-MBD and an apparently non-functional C-terminal MBD. The Cu+ dependent interaction of N-MBD and ATPBD was postulated as a possible mechanism for enzyme regulation. The Cu+ transfer from the chaperone to CopA is independent of the N-MBD capability to bind Cu+. Therefore, we hypothesized that ligand (Cu+ or nucleotide) binding to cytoplasmic domains might affect the interactions between the cytoplasmic domains. To test these ideas, the interactions among isolated cytoplasmic domains were characterized. Studies using isolated domains showed that while the N-MBD interacts with ATPBD, the presence of Cu+ or nucleotide (ADP) prevents this interaction. The N-MBD does not interact with the A domain. Alternatively, the C-MBD interacts with both ATPBD and A-domains in a ligand independent fashion. Only one Cu+ is transferred to CopA in absence of nucleotides, while the presence of ADP allows full loading of TM-MBS. These results suggest that the ligand binding affects the interactions between the cytoplasmic domains, and also change the conformation of CopA to help it accept the second Cu+ from chaperone.

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