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Assessment of the role of cannabinoid receptor 2 in innate immune cell trafficking during acute inflammationTaylor, Lewis January 2016 (has links)
Activation of the cannabinoid receptor CB<sub>2</sub> has been shown to induce directed leukocyte migration and inhibit leukocyte chemotaxis towards CC chemokines. However, the role that CB<sub>2</sub> plays in regulating macrophage chemotaxis remains understudied. Using a real-time chemotaxis assay and a panel of chemically diverse CB<sub>2</sub> agonists, I set out to examine whether CB<sub>2</sub> modulates primary macrophage chemotaxis. Of 14 agonists tested, only a subset acted as bona fide macrophage chemoattractants. Surprisingly, despite being pertussis toxin-sensitive, neither pharmacological inhibition nor genetic ablation of CB<sub>2</sub> had any effect on CB<sub>2</sub> agonist-induced macrophage chemotaxis. Furthermore, the activation of CB2 had no effect on CCL2 or CCL5- induced macrophage chemotaxis. Therefore, the activation of CB2 does not inhibit CC chemokine-induced macrophage migration and a non-CB<sub>1</sub>/CB<sub>2</sub>, G<sub>i/o</sub>-coupled GPCR must transduce CB2 agonist-induced macrophage chemotaxis. To identify the GPCR responsible, I examined primary murine macrophage GPCR expression and found that they express 124 non-sensory GPCRs. Functional screening of candidate receptors demonstrated that the putative cannabinoid receptors GPR18 and GPR55 and the lipid binding GPCRs LPAR1&5, CYSLTR1&2 and GPER1, were not responsible for CB<sub>2</sub> agonist-induced macrophage chemotaxis. Alongside, a ligand-directed virtual screen, combined with functional testing, uncovered a novel chemotaxis positive chemical scaffold. Importantly, compounds in this series containing a photoaffinity label retained activity and will aid in the identification of the target(s) responsible for CB<sub>2</sub> agonist-induced macrophage chemotaxis in future photocrosslinking experiments. Finally, I assessed whether CB2 controls innate immune cell recruitment in vivo using the zymosan-induced dorsal air pouch inflammation model and animals genetically deleted for CB<sub>2</sub>. I found that CB<sub>2</sub><sup>-/-</sup> mice had increased air pouch neutrophil and monocyte numbers, as well as pro-inflammatory mediators, during the acute inflammatory phase. Interestingly, mixed bone marrow chimera experiments demonstrated that lack of CB<sub>2</sub> specifically in the myeloid population is responsible for increased neutrophil trafficking. Therefore these data demonstrate that CB<sub>2</sub> acts to regulate neutrophil recruitment during the acute inflammatory response.
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An in vitro study of neutrophil chemotaxisTodd, Gail 14 April 2020 (has links)
When, at the beginning of 1972, my scientific attention was first drawn to the subject of cellular participation in the inflanunatory response, I was struck by the need for an understanding of the cellular and molecular mechanisms whereby blood leucocytes are attracted to an area of injury. The literature at that time contained good technical accounts of methods available for studying chemotaxis in vitro and many reports of diverse compounds of biological origin with attractant, or chemotactic, properties for motile, phagocytic cells. In general, these reports tended to substantiate the belief that chemical substances generated at an inflannnatory source attracted cells to that source in a teleologically appropriate way and they justified, by the consistent correlation observed, the relevance of in vitro procedures for studying the phenomenon. In other words, answers were available to the question "What substances attract?"; very few were available to the question, "How do they attract?".
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Quantification and functional characterization of Sinorhizobium meliloti chemotaxis proteinsArapov, Timofey Dmitryevich 19 March 2020 (has links)
The flagellated soil-dwelling bacterium Sinorhizobium meliloti is known for its symbiotic relationship with several leguminous genera. The symbiosis between the bacterium and its host plants facilitates fixation of atmospheric nitrogen and ultimately replenishment of nitrogen to the soil. However, before nitrogen fixation can occur, the bacterial cells must actively travel to the plant's roots and successfully induce formation of a plant organ called a root nodule. To initiate the nodulation process, the bacterium needs to be in direct contact with the root hairs. This requires free living bacterial cells within the soil to sense the presence of their host plant and travel to its roots. S. meliloti is able to do this through a process called chemotaxis. Chemotaxis is the ability to respond to chemical gradients within the environment by directed movement. It is facilitated by Methyl-accepting Chemotaxis Proteins (MCPs) as part of a two-component signal transduction system. These receptor proteins are able to bind ligands and influence the state of the signal transduction system, ultimately controlling flagellar behavior. The chemotaxis system of Escherichia coli has been well characterized and serves as a useful point of comparison to that of S. melilot throughout this work.
Within this work we have determined the stoichiometry of all chemotaxis proteins of S. meliloti by means of quantitative immunoblotting. Chapter 2 addresses the stoichiometry of MCPs and the histidine kinase CheA. The eight MCPs were grouped by total abundance within the cell, in high abundance (McpV), low abundance (IcpA, McpU, McpX, and McpW), and very low abundance (McpY, McpZ and McpT). The approximate cellular ratio of these three receptor groups is 300:30:1. The chemoreceptor-to-CheA ratio is 22.3:1, highly similar to the 23:1 ratio known for Bacilius subtiltis.
Chapter 3 continues the investigation of the protein stoichiometry, expanding to all chemotaxis proteins. We compare ratios of S. meliloti chemotaxis proteins to those of E. coli and B. subtilis. We address the possible reasons for the high ratio of CheR / CheB to the total amount of receptors. Proteins again can be grouped by abundance: CheD, CheY1, and CheY2 are the most abundant. CheR and CheB appear in lower amounts, CheS and CheT appear to be auxiliary proteins, and finally CheW1 and CheW2, which directly interact with the receptors and CheA.
Chapter 4 focuses on altered receptor abundance in S. meliloti due to the fusion of common epitope tags to the C-terminus. The fusion of these tags promotes greater cellular abundance of many receptors including McpU. The fusion of charged residues to the C-terminus promotes a greater increase in McpU abundance thanthe addition of single amino acid residues. Truncations of McpU were made to investigate the presence of a protease recognition site near the C-terminus. These truncations resulted in an increase in abundance similar to those resulting from epitope tag fusions. As epitope tags are widely used in protein studies to help determine protein stoichiometry, this study obviates a potential stumbling block for future experimenters.
The function of CheT, a small protein (13.4 kDa) encoded by the last gene in S. meliloti's major chemotaxis operon, is the subject of chapter 5. A cheT deletion strain is chemotactically deficient compared to the S. meliloti wild-type strain. Through two separate experiments (a glutaraldehyde cross-linking assay and co-purification) we demonstrated that CheT interacts with the methyltransferase CheR. We also investigate its possible role in CheR's methylation activity through a series of methylation assays.
This work contributes to our understanding of Sinorhizobium meliloti's chemotaxis signal transduction system. We have discovered evidence for new a protein-protein interaction within our system and have revealed the abundance of all chemotaxis proteins within the cell. We also showed that fusions of epitope tags to various chemotaxis proteins can dramatically influence their abundance. We shed light on the possible function of a previously uncharacterized protein, although more work is required to determine its exact role. / Doctor of Philosophy / Sinorhizboium meliloti is a bacterium that lives in the soil and forms a symbiotic relationship with many plants including alfalfa, a commonly grown cover crop. This symbiotic relationship is important because it allows for nitrogen to be replenished into the soil without the use of artificial fertilizer. However, to form this relationship the bacterial cells in the soil must be able to colonize the plant roots. The soil is a complex environment with many different kinds of chemical molecules and sources of nutrients. Like many other types of bacteria, S. meliloti uses flagella (long helical structures that rotate much like a propeller) to move through the soil. Control of the flagella falls to what is known as a chemotaxis signal transduction system, which can be thought of as a navigation system for each bacterial cell. The system has proteins that act as receptors to sense different chemical molecules. The bacterial cells can sense signals for the plant and move towards their host. This work shows the abundance of each type of receptor and other components within the cell. It also examines the function of a previously unknown protein, CheT, within the chemotaxis system.
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The Potential of Coelomocyte Chemotaxis as an Immune Biomarker in the Earthworm, Lumbricus terrestrisMota, Jennifer A. 12 1900 (has links)
Coelomocyte migration responses, both random and chemotatic, were examined in the earthworm Lumbricus terrestris. Coelomocyte random migration patterns towards non-stimulatory, non-chemotatic solutions were described. Migration responses to immunostimulatory agents lipopolysaccharides (LPS), N-formly-methionyl-leucyl-phenylalanine (FMLP), sheep erythrocytes, Saccharomyces cerevisiae, Aeromonas hydrophila, Eisenia fetida and Rhabditis pellio were characterized. Chemotaxis was reported to LPS, FMLP, sheep erythrocytes, S. cerivesae and E. fetida. Bio-indicator potential of chemotaxis is discussed relative to variability in migration responses.
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Characterization of the Chemotaxis System of the Endosymbiotic Bacterium Rhizobium leguminosarumMiller, Lance Delano 24 August 2007 (has links)
Chemotaxis is the process by which motile bacteria navigate chemical gradients in order to position themselves in optimum environments for growth and metabolism. Sensory input from both the external environment and the internal cellular environment are sensed by chemotaxis transducers and transduced to a two-component system whose output interacts with the flagellum thereby regulating motility.
Chemotaxis has been implicated in establishing the endosymbiotic relationship between the motile alpha-proteobacterium Rhizobium leguminosarum biovar viciae and its host Pisum sativa, the pea plant. An approach combing bioinformatical sequence analysis, molecular genetics, and behavioral analysis was used to characterize the chemotaxis system of R. leguminosarum and determine its contribution to this bacterium s lifestyle.
A genome search revealed the presence of two chemotaxis gene clusters, che1 and che2. Homologs of each che cluster are major chemotaxis operons controlling flagellar motility in other bacterial species. For this reason we sought to determine the contribution of each che cluster to chemotaxis in R. leguminosarum. We found that while both che clusters contribute to the regulation of motility, che1 is the major che cluster controlling chemotaxis. Using competitive nodulation assays we determined that che1, but not che2, is essential for competitive nodulation.
The major che cluster, che1, encodes a chemotaxis transducer, IcpA-Rl, with a globin coupled sensor domain. Chemotaxis transducers with a globin coupled sensor domain comprise a large class of proteins found in bacteria and archaea. These proteins have been shown to bind heme and sense oxygen and are therefore termed HemATs for heme-binding aerotaxis transducers. However, sequence analysis of IcpA-Rl reveals that it lacks the requisite amino acid residues for heme-binding and is therefore unlikely to sense oxygen. We present evidence that IcpA-Rl is likely an energy transducer and represents a novel function of the globin coupled sensor domain in sensing energy related parameters.
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Chemotactic activity of dental plaque a dissertation [sic] submitted in partial fulfillment ... periodontics /Kraal, Jan Hendrik. January 1972 (has links)
Thesis (M.S.)--University of Michigan, 1972.
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Measuring chemotaxis in Borrelia burgdorferi the Lyme disease spirocheteBakker, Richard Gerrit. January 1900 (has links)
Thesis (Ph. D.)--West Virginia University, 2004. / Title from document title page. Document formatted into pages; contains x, 138 p. : ill. (some col.). Vita. Includes abstract. Includes bibliographical references (p. 108-136).
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Chemotactic activity of dental plaque a dissertation [sic] submitted in partial fulfillment ... periodontics /Kraal, Jan Hendrik. January 1972 (has links)
Thesis (M.S.)--University of Michigan, 1972.
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Assortative Fertilization in the Elegans-Group of <i>Caenorhabditis</i>Seibert, Sara Rose January 2014 (has links)
No description available.
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Elucidation of the Specificity of S. meliloti Chemoreceptors for Host Derived AttractantsWebb, Benjamin A. 24 August 2016 (has links)
The bacterium Sinorhizobium (Ensifer) meliloti is a member of the Rhizobiaceae family and can enter a mutualistic, diazotrophic relationship with most plants of the genera Medicago, Melilotus, and Trigonella. Medicago sativa (alfalfa) is an agriculturally important legume that hosts S. meliloti and allows the bacterium to invade the plant root and begin fixing nitrogen. Prior to invasion, S. meliloti exists as a free living bacterium and must navigate through the soil to find alfalfa, using chemical signals secreted by the root. Alfalfa is the 4th most cultivated crop in the United States, therefore, identification of plant host signals that lure S. meliloti, and identification of the bacterium's chemoreceptors that perceive the signals can aid in propagating the symbiosis more efficiently, thus leading to greater crop yields. Investigations here focus on discovering alfalfa derived attractant signals and matching them to their respective chemoreceptors in S. meliloti. We have determined the chemotactic potency of alfalfa seed exudate and characterized and quantified two classes of attractant compounds exuded by germinating alfalfa seeds, namely, amino acids and quaternary ammonium compounds (QACs). At all points possible, we have compared alfalfa with the closely related non-host, spotted medic (Medicago arabica). The chemotactic potency of alfalfa seed exudate is the same as spotted medic seed exudate, however, the attractant compositions are chemically different. The amount of each proteinogenic amino acid (AA) exuded by spotted medic is slightly greater than the amounts exuded by alfalfa. In addition, the five QACs studied are exuded in various amounts between the two Medicago species. In comparison, the total amount of proteinogenic AAs exuded be alfalfa and spotted medic are 2.01 μg/seed and 1.94 μg/seed respectively, and the total amount of QACs exuded are 249 ng/seed and 221 ng/seed respectively. By performing a chemotaxis assay with synthetic AA mixtures mimicking the amounts exuded from the medics, it was found that the AA mixtures contribute to 23% and 37% of the responses to alfalfa and spotted medic exudates, respectively. The chemoreceptor McpU was found to be the most important chemoreceptor of the eight for chemotaxis to the whole exudates and the AA mixtures. Furthermore, McpU is shown to mediate chemotaxis to 19 of 20 AAs excluding aspartate. McpU directly interacts with 18 AAs and indirectly mediates chemotaxis to glutamate. Through single amino acid residue substitutions, it is determined that McpU directly binds to amino acids in the annotated region called the Cache_1 domain, likely utilizing residues D155 and D182 to interact with the amino group of AA ligands. In all, McpU is a direct sensor for AAs except for the acidic AAs aspartate and glutamate. Work is presented to show that the QACs betonicine, choline, glycine betaine, stachydrine, and trigonelline are potent attractants for S. meliloti, McpX is the most important chemoreceptor for chemotaxis to these QACs, and we demonstrate the binding strength of McpX to the QACs with dissociation constants ranging from low millimolar to low nanomolar, thus making McpX the first observed bacterial MCP that mediates chemotaxis to QACs. Overall, we match medic derived AAs with McpU and QACs with McpX. These results can aid in optimizing chemotaxis to the host derived attractants in order to propagate the symbiosis more efficiently resulting in greater crop yields.
Chapter 2 characterizes the function of the S. meliloti Methyl accepting Chemotaxis Protein U (McpU) as receptor for the attractant, proline. A reduction in chemotaxis to proline is observed in an McpU deletion strain, but the defect is restored in an mcpU complemented strain. Single amino acid substitution mutant strains were created, each harboring a mutant mcpU gene. The behavioral experiments with the mutants display a reduction in chemotaxis to proline when aspartate 155 and aspartate 182 are changed to glutamates. The periplasmic region of wild type McpU was purified and demonstrated to directly bind proline with a dissociation constant (Kd) of 104 μM. The variant McpU proteins show a reduction in binding affinity confirming McpU as a direct proline sensor.
Chapter 3, describes the development of a high-throughput technique that is able to observe chemotaxis responses in ten separate chemotaxis chambers all at once. This procedure also allows for real time observations at intervals of two minutes for however long the experiment is scheduled. Using this new method it was found that McpU and the Internal Chemotaxis Protein A (IcpA) are the most involved with chemotaxis to seed exudates followed by McpV, W, X, and Y. The amounts of each proteinogenic amino acid (AA) in host and non-host seed exudates are quantified, which reveals that similar amounts are exuded from each species. It is shown that McpU is the most important receptor for chemotaxis toward synthetic mixtures that mimic the amounts seen in the exudates.
Chapter 4 further investigates the role of McpU in sensing amino acids using the high-throughput technique developed in Chapter 3. It is shown that McpU is important for chemotaxis to all individual proteinogenic amino acids except the acidic AA, aspartate. In vitro binding experiments confirm that McpU directly interacts with all AAs except the acidic AAs aspartate and glutamate. Binding parameters are determined for aspartate, glutamate, phenylalanine and proline.
In Chapter 5, five quaternary ammonium compounds (QACs) are quantified from the host and non-host seed exudates, which reveals distinctive QAC profiles. S. meliloti is found to display strong chemotaxis to all QACs, which is further shown to be mediated mostly by McpX. McpX is then established as a direct binder to all QACs as well as proline, with dissociation constants ranging from nanomolar to millimolar.
These studies have increased our knowledge of how chemoreceptors sense attractants, and they have contributed to the bank of known attractant molecules for bacteria. Our new understandings of chemotaxis and how it relates to the Sinorhizobium-alfalfa model can allow for manipulations of the system to enhance chemotaxis to the host, thus propagating the symbiosis more efficiently, ultimately leading to greater crop yields. / Ph. D.
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