Suppression of ras-mediated transformation and inhibition of tumor growth and angiogenesis by anthrax lethal factor, a proteolytic inhibitor of multiple MEK pathways

Lethal factor is a protease, one component of Bacillus anthracis exotoxin, which cleaves many of the mitogen-activated protein kinase kinases (MEKs). Given the importance of MEK signaling in tumorigenesis, we assessed the effects of anthrax lethal toxin (LeTx) on tumor cells. LeTx was very effective in inhibiting mitogen-activated protein kinase activation in V12 H-ras-transformed NIH 3T3 cells. In vitro, treatment of transformed cells with LeTx caused them to revert to a nontransformed morphology, and inhibited their abilities to form colonies in soft agar and to invade Matrigel without markedly affecting cell proliferation. In vivo, LeTx inhibited growth of ras-transformed cells implanted in athymic nude mice (in some cases causing tumor regression) at concentrations that caused no apparent animal toxicity. Unexpectedly, LeTx also greatly decreased tumor neovascularization. These results demonstrate that LeTx potently inhibits ras-mediated tumor growth and is a potential antitumor therapeutic.

Growth factor treatment enhances vestibular hair cell renewal and results in improved vestibular function

The vestibules of adult guinea pigs were lesioned with gentamicin and then treated with perilymphatic infusion of either of two growth factor mixtures (i.e., GF I or GF II). GF I contained transforming growth factor α (TGFα), insulin-like growth factor type one (IGF-1), and retinoic acid (RA), whereas GF II contained those three factors and brain-derived neurotrophic factor. Treatment with GF I significantly enhanced vestibular hair cell renewal in ototoxin-damaged utricles and the maturation of stereociliary bundle morphology. The addition of brain-derived neurotrophic factor to the GF II infusion mixture resulted in the return of type 1 vestibular hair cells in ototoxin-damaged cristae, and improved vestibular function. These results suggest that growth factor therapy may be an effective treatment for balance disorders that are the result of hair cell dysfunction and/or loss.

Identification of a c-fos-induced gene that is related to the platelet-derived growth factor/vascular endothelial growth factor family.

Using a mRNA differential screening of fibroblasts differing for the expression of c-fos we isolated a c-fos-induced growth factor (FIGF). The deduced protein sequence predicts that the cDNA codes for a new member of the platelet-derived growth factor/vascular endothelial growth factor (PDGF/VEGF) family. Northern blot analysis shows that FIGF expression is strongly reduced in c-fos-deficient cells. Transfection of exogenous c-fos driven by a constitutive promoter restores the FIGF expression in these cells. In contrast, both PDGF and VEGF expression is unaffected by c-fos. FIGF is a secreted dimeric protein able to stimulate mitogenic activity in fibroblasts. FIGF overexpression induces morphological alterations in fibroblasts. The cells acquire a spindle-shaped morphology, become more refractive, disorganized, and detach from the plate. These results imply that FIGF is a downstream growth and morphogenic effector of c-fos. These results also suggest that the expression of FIGF in response to c-fos activation induces specific differentiation patterns and its aberrant activation contributes to the malignant phenotype of tumors.

Chronic morphine induces visible changes in the morphology of mesolimbic dopamine neurons.

The mesolimbic dopamine system, which arises in the ventral tegmental area (VTA), is an important neural substrate for opiate reinforcement and addiction. Chronic exposure to opiates is known to produce biochemical adaptations in this brain region. We now show that these adaptations are associated with structural changes in VTA dopamine neurons. Individual VTA neurons in paraformaldehyde-fixed brain sections from control or morphine-treated rats were injected with the fluorescent dye Lucifer yellow. The identity of the injected cells as dopaminergic or nondopaminergic was determined by immunohistochemical labeling of the sections for tyrosine hydroxylase. Chronic morphine treatment resulted in a mean approximately 25% reduction in the area and perimeter of VTA dopamine neurons. This reduction in cell size was prevented by concomitant treatment of rats with naltrexone, an opioid receptor antagonist, as well as by intra-VTA infusion of brain-derived neurotrophic factor. In contrast, chronic morphine treatment did not alter the size of nondopaminergic neurons in the VTA, nor did it affect the total number of dopaminergic neurons in this brain region. The results of these studies provide direct evidence for structural alterations in VTA dopamine neurons as a consequence of chronic opiate exposure, which could contribute to changes in mesolimbic dopamine function associated with addiction.

Cell acidification in apoptosis: granulocyte colony-stimulating factor delays programmed cell death in neutrophils by up-regulating the vacuolar H(+)-ATPase.

Neutrophils in tissue culture spontaneously undergo programmed cell death (apoptosis), a process characterized by well-defined morphological alterations affecting the cell nucleus. We found that these morphological changes were preceded by intracellular acidification and that acidification and the apoptotic changes in nuclear morphology were both delayed by granulocyte colony-stimulating factor (G-CSF). Among the agents that defend neutrophils against intracellular acidification is a vacuolar H(+)-ATPase that pumps protons out of the cytosol. When this proton pump was inhibited by bafilomycin A1, G-CSF no longer protected the neutrophils against apoptosis. We conclude that G-CSF delays apoptosis in neutrophils by up-regulating the cells' vacuolar H(+)-ATPase and that intracellular acidification is an early event in the apoptosis program.

Inhibitory Control is a Rate-Limiting Factor to Preschoolers' Use of Irregular Inflection

We investigated whether children’s inhibitory control is associated with their ability to produce irregular verb forms as well as learn from corrective feedback following their use of an over-regularized form. Forty-eight 3.5 to 4.5 year old children were tested on the irregular past tense and provided with adult corrective input via models of correct use or recasts of errors following ungrammatical responses. Inhibitory control was assessed with a three-item battery of tasks that required suppressing a prepotent response in favor of a non-canonical one. Results showed that inhibitory control was predictive of children’s initial production of irregular forms and not associated with their post-feedback production of irregulars. These findings show that children’s executive functioning skills may be a rate-limiting factor on their ability to produce correct forms, but might not interact with their ability to learn from input in this domain. Findings are discussed in terms of current theories of past-tense acquisition and learning from input more broadly.

The Effects of Postural Loading, Sacral Orientation, and Age on Sex Differences in Lumbar Functional Morphology and Health

Thesis (Ph.D.)--University of Washington, 2016-06

HMG box transcription factor gene Hbp1 is expressed in germ cells of the developing mouse testis

HMG box containing protein 1 (HBP1) is a high mobility group domain transcriptional repressor that regulates proliferation in differentiated tissues. We have found mouse Hbp1 to be expressed strongly in the embryonic mouse testis from approximately 12.5 days post coitum, compared with low levels of expression in the embryonic ovary. Expression of Hbp1 is maintained in the developing testis beyond the onset of spermatogenesis after birth. Whole-mount in situ hybridisation analysis showed that expression of Hbp1 in the XY gonad is localized within the developing testis cords, the precursors of the seminiferous tubules. Expression of Hbp1 is not apparent in testis cords of gonads from homozygous We mutant embryos, which lack germ cells. In situ hybridisation analysis on cryosectioned embryonic testis indicated that Hbp1 expression resembles that of the germ cell marker Oct4. We conclude that Hbp1 is up-regulated specifically in germ cells of the developing XY gonad. The expression of Hbp1 in XY germ cells appears to correlate with the onset of mitotic arrest in these cells. (C) 2004 Wiley-Liss, Inc.

Cosuppression of eukaryotic release factor 1-1 in Arabidopsis affects cell elongation and radial cell division

The role of the eukaryotic release factor 1 (eRF1) in translation termination has previously been established in yeast; however, only limited characterization has been performed on any plant homologs. Here, we demonstrate that cosuppression of eRF1-1 in Arabidopsis (Arabidopsis thaliana) has a profound effect on plant morphology, resulting in what we term the broomhead phenotype. These plants primarily exhibit a reduction in internode elongation causing the formation of a broomhead-like cluster of malformed siliques at the top of the inflorescence stem. Histological analysis of broomhead stems revealed that cells are reduced in height and display ectopic lignification of the phloem cap cells, some phloem sieve cells, and regions of the fascicular cambium, as well as enhanced lignification of the interfascicular fibers. We also show that cell division in the fascicular cambial regions is altered, with the majority of vascular bundles containing cambial cells that are disorganized and possess enlarged nuclei. This is the first attempt at functional characterization of a release factor in vivo in plants and demonstrates the importance of eRF1-1 function in Arabidopsis.

A nonantisense sequence-selective effect of a phosphorothioate oligodeoxynucleotide directed against the epidermal growth factor receptor in A431 cells

The overexpression of epidermal growth factor receptor (EGFr) has been implicated as a causative factor and a poor prognostic marker in a number of carcinomas. Therefore, strategies that down-regulate EGFr expression may be therapeutically useful. We designed antisense ODNs complementary to the initiation codon region of the EGFr mRNA and evaluated their efficacy in several tumor-derived cells, including the A431 cell line that express amplified levels of EGFr. A 15-mer phosphorothioate (PS) antisense ODN (erbB1AS15) induced a concentration-dependent reduction in proliferation that was accompanied by a change in the morphology of A431 cells into more tightly clustered and discrete colonies. A 15-mer sense (PS) control oligodeoxynucleotide (ODN) and a phosphodiester (PO) version of erbB1AS15 had little or no effect on cell number of morphology, and erbB1AS15 (PS) did not induce these effects in control cell lines expressing lower levels of EGFr. The effects of erbB1AS15 (PS) on A431 cells were not mediated by a true antisense mechanism in that there was no reduction in the level of EGFr mRNA or protein over a 24-hr period, as determined by Northern and Western blotting, respectively. However, autophosphorylation of the receptor was significantly reduced by erbB1AS15 (PS) and not by control ODNs. The results of further studies suggested that this effect was mediated by a direct, dose-dependent inhibition of the EGFr tyrosine kinase enzyme and was not due to impairment of either ligand-binding or receptor dimerization. These data suggest that erbB1AS15 (PS) can inhibit proliferation and alter the morphology of A431 cells by a sequence-selective, but nonantisense mechanism affecting receptor tyrosine kinase activity.

Factors determining the morphology of β-amyloid (Aβ) deposits in Down's syndrome

Immunostained preparations of the medial temporal lobe from patients with Down's syndrome (DS) were counterstained with cresyl violet to reveal the β-amyloid (Aβ) deposits and their associated cell populations. Aβ deposits in the cornu Ammonis (CA) of the hippocampus were, on average, more strongly stained, less often directly associated with neurons and more often associated with glial cells than the adjacent areas of cortex. Cored deposits were more frequently recorded in sulci rather than gyri and were associated with more glial cells than the uncored deposits. Multiple regression analyses suggested there was a positive correlation in the cortex between Aβ deposit size and the frequency of closely associated neurons, the correlation being most significant with larger (>25 μm) neurons. The morphology of Aβ deposit was also correlated with the location of deposits in the cortex, CA and dentate gyrus but this factor was of lesser importance. No significant variation in the morphology of the Aβ deposits was associated with the presence of blood vessels within or adjacent to the deposit. The data suggest that neuronal cell bodies are important in the initial formation of Aβ deposits and glial cells with the development of more mature amyloid deposits.

The Relationship of Heteroptera Species Richness, Abundance, and Morphology to Elevation Gradients and Land-use Regimes on Mt. Kilimanjaro

Insect biodiversity is unevenly distributed on local, regional, and global scales. Elevation is a key factor in the uneven distribution of insect diversity, serving as a proxy for a host of environmental variables. My study examines the relationship of Heteroptera (true bugs) species diversity, abundance, and morphology to elevational gradients and land-use regimes on Mt. Kilimanjaro, Tanzania, East Africa. Heteroptera specimens were collected from 60 research sites covering an elevational range of 3684m (866-4550m above sea level). Thirty of the sites were classified as natural, while the remaining 30 were classified as disturbed (e.g., agricultural use or converted to grasslands). I measured aspects of the body size of adult specimens and recorded their location of origin. I used regression models to analyze the relationships of Heteroptera species richness, abundance, and body measurements to elevation and land-use regime. Richness and abundance declined with greater elevation, controlling for land use. The declines were linear or logarithmic in form, depending on the model. Richness and abundance were greater in natural than disturbed sites, controlling for elevation. According to an interaction, richness decreased more in natural than disturbed sites with rising elevation. Body length increased as a quadratic function of elevation, adjusting for land use. Body width X length decreased as a logarithmic function of elevation, while leg length/body length decreased as a quadratic function. Leg length/body length was greater in disturbed than natural sites. Interactions indicated that body length and body width X length were greater in natural than disturbed sites as elevation rose, although the general trend was downward. Future research should examine the relative importance of land area, temperature, and resource constraints for Heteroptera diversity and morphology on Mt. Kilimanjaro.

Structure, Morphology, and Electrochemical Properties of Transition Metal Oxide, Hydroxide, and Phosphate Nanomaterials for Energy Storage

Energy storage technologies are crucial for efficient utilization of electricity. Supercapacitors and rechargeable batteries are of currently available energy storage systems. Transition metal oxides, hydroxides, and phosphates are the most intensely investigated electrode materials for supercapacitors and rechargeable batteries due to their high theoretical charge storage capacities resulted from reversible electrochemical reactions. Their insulating nature, however, causes sluggish electron transport kinetics within these electrode materials, hindering them from reaching the theoretical maximum. The conductivity of these transition metal based-electrode materials can be improved through three main approaches; nanostructuring, chemical substitution, and introducing carbon matrices. These approaches often lead to unique electrochemical properties when combined and balanced.

Ethanol-mediated solvothermal synthesis we developed is found to be highly effective for controlling size and morphology of transition metal-based electrode materials for both pseudocapacitors and batteries. The morphology and the degree of crystallinity of nickel hydroxide are systematically changed by adding various amounts glucose to the solvothermal synthesis. Nickel hydroxide produced in this manner exhibited increased pseudocapacitance, which is partially attributed to the increased surface area. Interestingly, this morphology effect on cobalt doped-nickel hydroxide is found to be more effective at low cobalt contents than at high cobalt contents in terms of improving the electrochemical performance.

Moreover, a thin layer of densely packed nickel oxide flakes on carbon paper substrate was successfully prepared via the glucose-assisted solvothermal synthesis, resulting in the improved electrode conductivity. When reduced graphene oxide was used for conductive coating on as-prepared nickel oxide electrode, the electrode conductivity was only slightly improved. This finding reveals that the influence of reduced graphene oxide coating, increasing the electrode conductivity, is not that obvious when the electrode is already highly conductive to begin with.

We were able to successfully control the interlayer spacing and reduce the particle size of layered titanium hydrogeno phosphate material using our ethanol-mediated solvothermal reaction. In layered structure, interlayer spacing is the key parameter for fast ion diffusion kinetics. The nanosized layered structure prepared via our method, however, exhibited high sodium-ion storage capacity regardless of the interlayer spacing, implying that interlayer space may not be the primary factor for sodium-ion diffusion in nanostructured materials, where many interstitials are available for sodium-ion diffusion.

Our ethanol-mediated solvothermal reaction was also effective for synthesis of NaTi2(PO4)3 nanoparticles with uniform size and morphology, well connected by a carbon nanotube network. This composite electrode exhibited high capacity, which is comparable to that in aqueous electrolyte, probably due to the uniform morphology and size where the preferable surface for sodium-ion diffusion is always available in all individual particles.

Fundamental understandings of the relationship between electrode microstructures and electrochemical properties discussed in this dissertation will be important to design high performance energy storage system applications.

Inhibitory Control is a Rate-Limiting Factor to Preschoolers' Use of Irregular Inflection

We investigated whether children’s inhibitory control is associated with their ability to produce irregular verb forms as well as learn from corrective feedback following their use of an over-regularized form. Forty-eight 3.5 to 4.5 year old children were tested on the irregular past tense and provided with adult corrective input via models of correct use or recasts of errors following ungrammatical responses. Inhibitory control was assessed with a three-item battery of tasks that required suppressing a prepotent response in favor of a non-canonical one. Results showed that inhibitory control was predictive of children’s initial production of irregular forms and not associated with their post-feedback production of irregulars. These findings show that children’s executive functioning skills may be a rate-limiting factor on their ability to produce correct forms, but might not interact with their ability to learn from input in this domain. Findings are discussed in terms of current theories of past-tense acquisition and learning from input more broadly.

Temperature affects the morphology and calcification of Emiliania huxleyi strains

The global warming debate has sparked an unprecedented interest in temperature effects on coccolithophores. The calcification response to temperature changes reported in the literature, however, is ambiguous. The two main sources of this ambiguity are putatively differences in experimental setup and strain specificity. In this study we therefore compare three strains isolated in the North Pacific under identical experimental conditions. Three strains of Emiliania huxleyi type A were grown under non-limiting nutrient and light conditions, at 10, 15, 20 and 25 °C. All three strains displayed similar growth rate versus temperature relationships, with an optimum at 20–25 °C. Elemental production (particulate inorganic carbon (PIC), particulate organic carbon (POC), total particulate nitrogen (TPN)), coccolith mass, coccolith size, and width of the tube element cycle were positively correlated with temperature over the sub-optimum to optimum temperature range. The correlation between PIC production and coccolith mass/size supports the notion that coccolith mass can be used as a proxy for PIC production in sediment samples. Increasing PIC production was significantly positively correlated with the percentage of incomplete coccoliths in one strain only. Generally, coccoliths were heavier when PIC production was higher. This shows that incompleteness of coccoliths is not due to time shortage at high PIC production. Sub-optimal growth temperatures lead to an increase in the percentage of malformed coccoliths in a strain-specific fashion. Since in total only six strains have been tested thus far, it is presently difficult to say whether sub-optimal temperature is an important factor causing malformations in the field. The most important parameter in biogeochemical terms, the PIC : POC ratio, shows a minimum at optimum growth temperature in all investigated strains. This clarifies the ambiguous picture featuring in the literature, i.e. discrepancies between PIC : POC–temperature relationships reported in different studies using different strains and different experimental setups. In summary, global warming might cause a decline in coccolithophore's PIC contribution to the rain ratio, as well as improved fitness in some genotypes due to fewer coccolith malformations.