Multiple genetic modifications of the erythromycin polyketide synthase to produce a library of novel “unnatural” natural products

The structures of complex polyketide natural products, such as erythromycin, are programmed by multifunctional polyketide synthases (PKSs) that contain modular arrangements of functional domains. The colinearity between the activities of modular PKS domains and structure of the polyketide product portends the generation of novel organic compounds—“unnatural” natural products—by genetic manipulation. We have engineered the erythromycin polyketide synthase genes to effect combinatorial alterations of catalytic activities in the biosynthetic pathway, generating a library of >50 macrolides that would be impractical to produce by chemical methods. The library includes examples of analogs with one, two, and three altered carbon centers of the polyketide products. The manipulation of multiple biosynthetic steps in a PKS is an important milestone toward the goal of producing large libraries of unnatural natural products for biological and pharmaceutical applications.

Fibroblast growth factors: An epigenetic mechanism of broad spectrum resistance to anticancer drugs

Based on the observation that removal of tumors from metastatic organs reversed their chemoresistance, we hypothesized that chemoresistance is induced by extracellular factors in tumor-bearing organs. By comparing chemosensitivity and proteins in different tumors (primary vs. metastases) and different culture systems (tumor fragment histocultures vs. monolayer cultures derived from the same tumor), we found elevated levels of acidic (aFGF) and basic (bFGF) fibroblast growth factors in the conditioned medium (CM) of solid and metastatic tumors. These CM induced broad spectrum resistance to drugs with diverse structures and action mechanisms (paclitaxel, doxorubicin, 5-fluorouracil). Inhibition of bFGF by mAb and its removal by immunoprecipitation resulted in complete reversal of the CM-induced chemoresistance, whereas inhibition/removal of aFGF resulted in partial reversal. Using CM that had been depleted of aFGF and/or bFGF and subsequently reconstituted with respective human recombinant proteins, we found that bFGF but not aFGF induced chemoresistance whereas aFGF amplified the bFGF effect. aFGF and bFGF fully accounted for the CM effect, indicating these proteins as the underlying mechanism of the chemoresistance. The FGF-induced resistance was not due to reduced intracellular drug accumulation or altered cell proliferation. We further showed that an inhibitor of aFGF/bFGF (suramin) enhanced the in vitro and in vivo activity of chemotherapy, resulting in shrinkage and eradication of well established human lung metastases in mice without enhancing toxicity. These results indicate elevated levels of extracellular aFGF/bFGF as an epigenetic mechanism by which cancer cells elude cytotoxic insult by chemotherapy, and provide a basis for designing new treatment strategies.

A second-generation vaccine protects against the psychoactive effects of cocaine

The effects of immunization with the second-generation cocaine immunoconjugate GND-keyhole limpet hemocyanin (KLH) or with the anti-cocaine mAb GNC92H2 were assessed in a model of acute cocaine-induced locomotor activity. After i.p. administration of cocaine⋅HCl (15 mg/kg), rats were tested in photocell cages, and stereotypy was rated to determine preimmunization drug response (baseline). Experimental animals were subjected to an immunization protocol with GND-KLH or treated with the mAb GNC92H2. Rats were then challenged with systemic cocaine, and their locomotor responses were again measured. Active immunization with GND-KLH produced a 76% decrease in the ambulatory measure (crossovers) in the experimental group and a 12% increase in the control group compared with baseline values. Also, stereotypic behavior was significantly suppressed in the vaccinated animals. Decreases in both measures were seen in the experimental group on two subsequent challenges. The maximum effect was observed at the time of the second challenge with a dramatic 80% decrease in crossovers. Treatment with GNC92H2 resulted in a 69% decrease in crossovers compared with baseline. This effect persisted across two additional challenges over 11 days with decreases of 46–47%. In contrast, the control group showed increases of up to 28%. Significant differences between groups were observed in the stereotypic measure in all three challenges. The results indicate that these immunopharmacotherapeutic agents have significant cocaine-blockade potential and therefore may offer an effective strategy for the treatment of cocaine abuse.

Endothelial cell surface F1-FO ATP synthase is active in ATP synthesis and is inhibited by angiostatin

Angiostatin blocks tumor angiogenesis in vivo, almost certainly through its demonstrated ability to block endothelial cell migration and proliferation. Although the mechanism of angiostatin action remains unknown, identification of F1-FO ATP synthase as the major angiostatin-binding site on the endothelial cell surface suggests that ATP metabolism may play a role in the angiostatin response. Previous studies noting the presence of F1 ATP synthase subunits on endothelial cells and certain cancer cells did not determine whether this enzyme was functional in ATP synthesis. We now demonstrate that all components of the F1 ATP synthase catalytic core are present on the endothelial cell surface, where they colocalize into discrete punctate structures. The surface-associated enzyme is active in ATP synthesis as shown by dual-label TLC and bioluminescence assays. Both ATP synthase and ATPase activities of the enzyme are inhibited by angiostatin as well as by antibodies directed against the α- and β-subunits of ATP synthase in cell-based and biochemical assays. Our data suggest that angiostatin inhibits vascularization by suppression of endothelial-surface ATP metabolism, which, in turn, may regulate vascular physiology by established mechanisms. We now have shown that antibodies directed against subunits of ATP synthase exhibit endothelial cell-inhibitory activities comparable to that of angiostatin, indicating that these antibodies function as angiostatin mimetics.

Subcutaneous vaccination with irradiated, cytokine-producing tumor cells stimulates CD8+ cell-mediated immunity against tumors located in the "immunologically privileged" central nervous system.

Vaccination with cytokine-producing tumor cells generates potent immune responses against tumors outside the central nervous system (CNS). The CNS, however, is a barrier to allograft and xenograft rejection, and established tumors within the CNS have failed to respond to other forms of systemic immunotherapy. To determine what barriers the "immunologically privileged" CNS would pose to cytokine-assisted tumor vaccines and what cytokines would be most efficacious against tumors within the CNS, we irradiated B16 murine melanoma cells producing murine interleukin 2 (IL-2), IL-3, IL-4, IL-6, gamma-interferon, or granulocyte-macrophage colony stimulating factor (GM-CSF) and used these cells as subcutaneous vaccines against tumors within the brain. Under conditions where untransfected B16 cells had no effect, cells producing IL-3, IL-6, or GM-CSF increased the survival of mice challenged with viable B16 cells in the brain. Vaccination with B16 cells producing IL-4 or gamma-interferon had no effect, and vaccination with B16 cells producing IL-2 decreased survival time. GM-CSF-producing vaccines were also able to increase survival in mice with pre-established tumors. The response elicited by GM-CSF-producing vaccines was found to be specific to tumor type and to be abrogated by depletion of CD8+ cells. Unlike the immunity generated against subcutaneous tumors by GM-CSF, however, the effector responses generated against tumors in the CNS were not dependent on CD4+ cells. These data suggest that cytokine-producing tumor cells are very potent stimulators of immunity against tumors within the CNS, but effector responses in the CNS may be different from those obtained against subcutaneous tumors.

Presynaptic association of Rad51 protein with selected sites in meiotic chromatin.

Eukaryotic homologs of Escherichia coli Rec-A protein have been shown to form nucleoprotein filaments with single-stranded DNA that recognize homologous sequences in duplex DNA. Several recent reports in four widely diverse species have demonstrated the association of RecA homologs with meiotic prophase chromatin. The current immunocytological study on mouse spermatocytes and oocytes shows that a eukaryotic homolog, Rad5l, associates with a subset of chromatin sites as early as premeiotic S phase, hours before either the appearance of precursors of synaptonemal complexes or the initiation of synapsis. When homologous chromosomes do begin to pair, the Rad5l-associated sequences are sites of initial contact between homologues and of localized DNA synthesis. Distribution of Rad5l foci on the chromatin of fully synapsed bivalents at early pachynema corresponds to an R-band pattern of mitotic chromosomes. R-bands are known to be preferred sites of both synaptic initiation and recombination. The time course of appearance of Rad51 association with chromatin, its distribution, and its interaction with other Rad5l-associated sequences suggests that it plays an important role preselection of sequences and synaptic initiation.

Mammalian ubiquitin-conjugating enzyme Ubc9 interacts with Rad51 recombination protein and localizes in synaptonemal complexes.

Hsubc9, a human gene encoding a ubiquitin-conjugating enzyme, has been cloned. The 18-kDa HsUbc9 protein is homologous to the ubiquitin-conjugating enzymes Hus5 of Schizosaccharomyces pombe and Ubc9 of Saccharomyces cerevisiae. The Hsubc9 gene complements a ubc9 mutation of S. cerevisiae. It has been mapped to chromosome 16p13.3 and is expressed in many human tissues, with the highest levels in testis and thymus. According to the Ga14 two-hybrid system analysis, HsUbc9 protein interacts with human recombination protein Rad51. A mouse homolog, Mmubc9, encodes an amino acid sequence that is identical to the human protein. In mouse spermatocytes, MmUbc9 protein, like Rad51 protein, localizes in synaptonemal complexes, which suggests that Ubc9 protein plays a regulatory role in meiosis.

Zubiri y Ellacuría: fundamentos filosóficos para una teoría estructural y una praxeología crítica de las relaciones internacionales

La hipótesis de partida de esta tesis es que la teoría de las Relaciones Internacionales (RRII) se encuentra ante un problema de carácter filosófico que impide su adecuada fundamentación. El objeto mismo de la teoría de las RRII ha sido foco de numerosas disputas a lo largo y ancho de los debates con que la disciplina ha explicado sus diversos posicionamientos y enfoques. De igual manera, diferentes aproximaciones metodológicas han ido evolucionando al albur de los avances que la Filosofía de la Ciencia venía incorporando. Así, los diversos puntos de vista han tratado de adaptarse a las novedades de carácter epistemológico que incorporaba la Teoría de la Ciencia en forma de nuevos paradigmas y programas de investigación sin detenerse lo suficiente en revisar los postulados sustanciales sobre los que partió y se desarrollaron las distintas teorizaciones. Con demasiada frecuencia, los conceptos que guiaron los esfuerzos de la revisión de dichos postulados fueron utilizados de forma superficial, tratando con ello más bien de sustentar las teorías pre-configuradas que de realizar una aproximación reflexiva a la realidad sobre la que se pretendía teorizar, lo que ha evidenciado una insuficiencia conceptual de la teoría de las RRII para hacer frente a la realidad (Ashley 1984; Bohman 2009; Cox y Schechter 2002; Jackson 2008; Lapid 2003; Monteiro y Ruby 2009; Walker 1993)...

The Clinical Utility of the Adult Attachment Projective Picture System: A Clinician's Perspective

The Adult Attachment Projective Picture System (AAP) is the first performance- based measure of adult attachment to be developed. The purpose of the measure is to provide a clinical understanding of an adult client's attachment status and associated coping mechanisms. The AAP is a relatively new measure that has yet to be examined from a utility perspective. In the current study, seven psychologists completed a structured survey in order to identify their perspectives of the AAP and its utility as a clinical instrument. A phenomenological qualitative analysis of the data was conducted to derive themes about the AAP and its clinical utility. Analyses aimed to answer the following: What clinical considerations do clinician's focus on when deciding to use this measure? What are common factors among clinician's who do use the measure as well as those who do not? What aspects of the measure are user-friendly and what aspects are difficult? General themes that emerged include (a) the clinical information provided by the AAP is viewed by those who use it as unique and beneficial; (b) time commitment and cost for the clinician are common considerations when clinician's are deciding whether or not to use the AAP or when pursuing training; (c) the AAP provides an increased understanding of one's relational capacities and defenses; and (d) the coding system and transcription process are difficult aspects of the AAP and influence how and/or when it is used. In addition to these themes, multiple respondents discussed potential changes for the AAP that would increase their future use of the instrument. Finally, the implications of these results are discussed.

Looking Back: A Granddaughter's Quest for Redemption and Understanding

On December 7th, 1941 Japan attacked Pearl Harbor in Hawaii. This event signaled the beginning of America's involvement in World War II. It also signaled the beginning of a change of life for the Japanese living in the United States. Following the attack, 120,000 persons of Japanese ancestry were placed in ten internment camps across the United States. Two-thirds of those interned were American citizens, including my grandmother. The fact that these American citizens were treated as prisoners in their own country had a devastating effect on the relationships of those interned and the generations of Japanese Americans that followed. This essay recounts my grandma's experience and explores how her internment changed her life and the lives of her descendants.

A Case Study of the Economic and Environmental Impacts from a Bio-fuel Facility (Southwest Georgia Ethanol, LLC) on the Surrounding Communities, Southwest Georgia

Bio-fuels such as ethanol provide an extraordinary opportunity to address our dependency on foreign oil. This case study examines the economic and environmental impacts associated with constructing and operating a dry mill ethanol manufacturing facility in a Southwest Georgia town and surrounding communities. The case study found that the plant had little impact on air quality, water quality, and habitat fragmentation. However, economic results showed the plant produced $1.5 million in tax revenues, and 86 jobs. Ethanol producers and communities must consider both the economic and environmental impacts on a local community when searching or attracting a bio-fuels plant. Likewise, communities should be aware of these challenges when attracting ethanol production plants.

Transcending Cultural and Language Differences: Ethical Codes of Conduct for Translators and Interpreters in Afghanistan

This project examines what an ethical code of conduct would look like in Afghanistan through analysis of historical, cultural and linguistic aspects found within its regions, as well as an examination of ethical codes of conduct for translators and interpreters in other countries. While numerous ethical guidelines and codes of conduct for translators and interpreters exist throughout global communities, it seems that creating a successful standardized ethical code of conduct in Afghanistan may be difficult to achieve given cultural and linguistic complexities. An ethical code of conduct for translators and interpreters in Afghanistan should include particular focus on: cultural sensitivity and courtesy, impartiality, conflict of interest, options for withdrawal, as well as reflect the importance of Pashtunwali.

Employee Leadership Development

Abstract \ The business importance of retaining and developing top performing employees is more crucial to business organizations that in the past, yet many organizations are not confident in their ability to staff leadership positions within the next five years. Many companies continue to run without a employee leadership development program. Organizations that shy away from development programs tend to do so because of fear the costs are too high. Organizations that support leadership programs acknowledge that the benefits far outweigh the costs. In this capstone study, the researcher illustrates that employee leadership development programs add value through improved business strategy and employee satisfaction, and as a result, represent a worthy investment.