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Interaction of Heparan Sulfate with Pro- and Anti-Angiogenic ProteinsVanwildemeersch, Maarten January 2006 (has links)
<p>Heparan sulfate (HS) is an unbranched and negatively charged polysaccharide of the glycosaminoglycan family, based on the repeated (GlcNAcα1-4GlcAβ1-4)<sub> </sub>disaccharide structure. The HS backbone is modified by epimerization and sulfation in various positions. HS chains are composed of <i>N</i>-sulfated (NS) domains – predominant locations for further modification steps –, the poorly modified <i>N</i>-acetylated (NA) domains and the alternating NA/NS-domains. HS is present at the cell surface and in the extra-cellular matrix and interacts at these sites with various proteins involved in numerous biological processes, such as angiogenesis. Both pro- and anti-angiogenic proteins can interact with HS and this study was focused on how HS binds to the anti-angiogenic proteins endostatin (ES) and histidine-rich glycoprotein (HRGP) and to pro-angiogenic fibroblast growth factors (FGFs).</p><p>Here we show that ES recognizes NS-domains in HS spaced by NA-disaccharides, and that binding to ES is abolish through cleavage at these NA-disaccharides. HRGP335, a peptide derived from the His/Pro-rich domain of HRGP is shown to bind to heparin and HS to the same extent as full-size HRGP, in a Zn<sup>2+</sup>-dependent manner. Moreover, the ability of HRGP to inhibit endothelial cell migration is located to the same region of the protein. We analyzed HS structure in respect to binding to HRGP335 and FGF-2, and show that the ability of HS to bind to those proteins depends on chain length and composition. Finally, the role of HS in FGF–HS–FGF receptor ternary complexes is evaluated using biosynthetic analogs of NS-domains. For stabilization of such complexes the overall sulfation degree of HS seems to play a more pronounced role than the exact distribution of sulfate groups.</p><p>The results presented in this thesis contribute to a greater understanding of the role of HS in angiogenesis and may provide valuable information for the development of cures against angiogenesis-related disorders.</p>
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Identification of PHPT1 in mouse tissues by immunohistochemistryKoria, Muntaha January 2007 (has links)
<p>Although it has been estimated that protein histidine phosphorylation account for about 6 % of the protein phosphorylation in eukaryotic cells; the knowledge of histidine phosphorylation and dephosphorylation is still limited. Lately, studies have appeared of a mammalian 14-kDa phospho- histidine phosphatase, also named protein histidine phosphatase and molecular cloning have provided some information of its physiological role. The object of the present study was to detect the protein expression of protein histidine phosphatase, PHPT1, in mouse tissue, by using immunohistochemistry. Tissue samples from a 4-week-old mouse (heart, liver, kidney, lung, muscle, and spleen), 5-month-old mouse (testis and intestinal), 8-month-old mouse (uterus) and an embryo from 14.5 days old mouse were obtained and processed for light microscopic examination. An absorption test was also made to confirm the specificity of the antibody. The results reveal that PHPT1 is mainly expressed in epithelium, heart- and skeletal muscle. These results provide new evidences for the understanding of the function of eukaryotic histidine phosphorylation and dephosphorylation.</p><p>KEYWORDS</p><p>Phosphohistidine, dephosphorylation, protein histidine phosphatase, phosphohistidine phosphatase, protein phosphorylation</p>
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Interaction of Heparan Sulfate with Pro- and Anti-Angiogenic ProteinsVanwildemeersch, Maarten January 2006 (has links)
Heparan sulfate (HS) is an unbranched and negatively charged polysaccharide of the glycosaminoglycan family, based on the repeated (GlcNAcα1-4GlcAβ1-4) disaccharide structure. The HS backbone is modified by epimerization and sulfation in various positions. HS chains are composed of N-sulfated (NS) domains – predominant locations for further modification steps –, the poorly modified N-acetylated (NA) domains and the alternating NA/NS-domains. HS is present at the cell surface and in the extra-cellular matrix and interacts at these sites with various proteins involved in numerous biological processes, such as angiogenesis. Both pro- and anti-angiogenic proteins can interact with HS and this study was focused on how HS binds to the anti-angiogenic proteins endostatin (ES) and histidine-rich glycoprotein (HRGP) and to pro-angiogenic fibroblast growth factors (FGFs). Here we show that ES recognizes NS-domains in HS spaced by NA-disaccharides, and that binding to ES is abolish through cleavage at these NA-disaccharides. HRGP335, a peptide derived from the His/Pro-rich domain of HRGP is shown to bind to heparin and HS to the same extent as full-size HRGP, in a Zn2+-dependent manner. Moreover, the ability of HRGP to inhibit endothelial cell migration is located to the same region of the protein. We analyzed HS structure in respect to binding to HRGP335 and FGF-2, and show that the ability of HS to bind to those proteins depends on chain length and composition. Finally, the role of HS in FGF–HS–FGF receptor ternary complexes is evaluated using biosynthetic analogs of NS-domains. For stabilization of such complexes the overall sulfation degree of HS seems to play a more pronounced role than the exact distribution of sulfate groups. The results presented in this thesis contribute to a greater understanding of the role of HS in angiogenesis and may provide valuable information for the development of cures against angiogenesis-related disorders.
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Ph Responsive Nano Carriers For Anti Cancer Drug DeliveryBagherifam, Shahla 01 March 2013 (has links) (PDF)
In the recent years, development of various organic and inorganic nano-sized systems has gained great interests especially for cancer diagnosis and treatment and intense researches are carried out in this area. Regarding to the recent trends for drug delivery system design, the novel approaches for drug carriers are mainly based on development of smart and nano-size drug carriers which are targeted to cancer cells. Hence, for an effective tumor-targeted delivery device, besides its chemical structure further criteria such as detection of tumor site and sensitivity to the higher temperature and lower pH of the tumor compare to rest of the body gains importance. The aim of this study is to design and prepare polysebacic anhydride (PSA) based nanocapsules (NCs) loaded with Doxorubicin (DOX) which is an anti cancer drug. In order to obtain an intelligent delivery system, drug-loaded nanocapsules were coated with pH sensitive poly (L-histidine). PSA nano-carriers were firstly loaded with DOX and then in order to introduce pH sensitivity, they were coated with poly (L-histidine). PLH-coated NCs were modified with polyethylene glycol (PEG) to prevent their macrophage uptake. Drug release profile from this system was examined in two different buffer solutions prepared as acidic (pH 4) and physiological (pH 7.4) media. The physical and chemical properties of the nano particles were characterized by Fourier transform infrared spectroscopy (FTIR), dynamic light scattering (DLS), ultraviolet and visible absorption spectroscopy (UV-VIS), and scanning electron microscopy (SEM). In vitro studies of the prepared nanocapsules were performed on MDA-MB-231 breast cancer cells by using WST Kit 8 cell viability test. In order to obtained results, pH sensitive nanocapsules with size 230 nm exhibited cellular uptake and promising intracellular release of drug.
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Preperation And Characterization Of Silica Coated Magnetite Nanoparticles And Labeling With Nonradioactive Re As A Surrogate Of Tc-99m For Magneticly Targeted ImagingZengin, Umit 01 December 2010 (has links) (PDF)
Magnetic nanoparticles have been used in many areas owing to their variable characteristic behaviors. Among these iron oxide nanoparticles are one of the mostly preferred type of nanoparticles. In this study Fe3O4, namely magnetite, which is one type of magnetic iron oxide nanoparticles was used. Magnetite nanoparticles with a narrow size distribution were prepared in aqueous solution using the controlled coprecipitation method. They were characterized by electron microscopic methods (SEM and TEM), crystal structure analysis (XRD), particle size analyzer, vibrating sample magnetometer (VSM) and Raman spectrometry. The nanoparticles were coated with a thin (ca 20 nm) silica shell utilizing the hydrolysis and the polycondensation of tetraethoxysilane (TEOS) under alkaline conditions in ethanol. The presence of silica coating was investigated by energy dispersive X-ray spectrometer (EDX) measurement. After surface modification with an amino silane coupling agent, (3-Aminopropyl)triethoxysilane, histidine was covalently linked to the amine group using glutaraldehyde as cross-linker. Carbonyl complexes of rhenium [Re(CO)3(H2O)3]+ was prepared through reductive carboxylation utilyzing gaseous carbon monoxide as a source of carbonyl and amine borane (BH3NH3) as
the reducing agent. The complex formation was followed by HPLC- ICP-MS system and 95% conversion of perrhanete into the complex was achieved. The magnetic nanoparticles were then labeled with the Re complex with a yield of 86.8% through the replacement of labile H2O groups with imidazolyl groups. Thus prepared particles were showed good stability in vitro. Herein rhenium was selected as a surrogate of radioactive 99mTc. However radioactive isotopes of rhenium (186-Re and 188 Re) is also used for radioactive therapy.
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Nuclear Behaviour In Heterokaryons : Genetic And Molecular Analysis Of (his-3+ his-3+) Heterokaryons Of Neurospora CrassaPitchairnani, K 06 1900 (has links)
In contrast to plant and animal cells, the fungal cells are multinucleate. A consequence of their multinucleate condition is heterokaryosis — the occurrence of genetically different nuclei in a common cytoplasm. In nature this condition occurs because of spontaneous mutations in the haploid nuclei in the coenocytic mycelium. Inspite of heterokaryosis being a fundamental aspect of fungal biology, the behaviour and dynamics of nuclei in fungal mycelium are little understood. This study was prompted by the following questions: (1) Why does a fungus need so many nuclei? (2) Are they all active simultaneously? (3) Does the proportion of the different nuclear types in fungal mycelium alter in response to change in conditions of growth? (4) Is the activity of an enzyme related to the dose of nuclei containing the encoding gene?
Experimental approach. The approach taken was to generate heterokaryons in which one of the nuclear types carries a mutant allele for a specific enzyme while the other nuclear type carries the functional allele, introduced by transformation. Because in filamentous fungi, the transforming DNA commonly integrates randomly into the chromosomal DNA, the transformants would be genetic 'variants' in which the ratios of transformed to non-transformed nuclei might be controlled differently. The transformants could thus be useful in investigating the relationship between the frequency of transformed nuclei and the activity of encoded enzyme. In addition the transformants might be useful for studying nuclear behaviour. The availability of developmental information, genetic and molecular methodology, and biochemical mutant in Neurospora crassa made this fungus a material of choice for this investigation.
Strain construction. A histidinol dehydrogenase (his-3) mutant strain was used into which an albino colour marker and a biochemical marker, inositoL were introduced by crossing. The latter two markers served as check against possible laboratory contamination. In addition, a gene mem, was introduced into the strain. In the mem genetic background, the strain has a wild-type morphology on agar medium but when grown in liquid shake culture it produces uninucleate microconidia that are useful in estimating nuclear ratio. Protoplasts of a constructed strain (his-3 al-1; mem; inl) were transformed with a plasmid containing the wild-type his-3 allele, thereby converting the original strain into a heterokaryotic strain having a mixture of transformed (his-3+t) and untransformed (his-3) nuclei. [The superscript +/ is used here to denote an his~3+ allele ectopically introduced by transformation]. Integration of plasmid DNA sequence in three selected transformants, 2T5, 3T3 and 4T12, was confirmed by genomic Southern analysis using the vector DNA as probe. The exponential growth rate of all three transformants was similar (~0.08mgh"1).
Nuclear ratio. Assuming a uniform distribution of nuclei in mycelium, and a correspondence between nuclear ratio in mycelium and conidia, the ratio his-3* {: his-3 was estimated by plating microconidia. In transformant 3T3, the nuclear ratio was 7:1. In 2T5, all nuclei were his-3n. Transformant 4T12 did not produce microconidia. The nuclear ratio in this transformant was therefore estimated by macroconidial plating and found to be 1:5, in favour of his-3 nuclei.
Behaviour of transformants in vegetative and sexual phase. Although the transformants had originally been selected for the expression of his-3+T gene, a majority of macroconidia produced in cultures of 3T3 and 2T5 required histidine to trigger their germination. This condition, referred to as cphenotypic lag', led to a gross underestimation of the proportion of prototrophic macroconidia by the direct plating method and biased the estimation of nuclear ratios. Therefore nuclear ratio was estimated by first germinating macroconidia on histidine supplemented medium before testing colonies in histidine dropout slants and comparing the numbers of auxotrophic and prototrophic mycelia. Phenotypic lag was not observed in 4T12. The variation in the degree of expression of phenotypic lag among the transformants was ascribed to transgene position effect. The transformants differed also in meiotic instability of the transforming DNA — the transforming DNA in 3T3 was passed through unchanged but it was deleted or modified in4T12and2T5.
Experimental alteration of nuclear ratio. The transformants differed with respect to the self-adjusted ratio of transformed to non-transformed nuclei and also to the degree to which their nuclear ratio could be altered by nutritional manipulation of the growth medium, i.e., by growing the transformants in the presence or absence of histidine in the medium. In 3T3, the proportion of his-3+t nuclei progressively decreased by 3.5-fold in the sixth subculture on histidine medium. The change in 4T12 was even more striking: in the sixth serial subculture, the proportion of his-3+t nuclei decreased from 17-20% to -0.05%.However, when it was propagated again in medium that lacked histidine, the frequency of his-3+t nuclei was immediately restored to original level (-17%). That drastic alterations in nuclear ratio occurred upon nutritional manipulation was verified by Southern analysis. The intensity of signal specific for transformed DNA (nuclei) in cultures grown without histidine supplement was strong, but barely detectable in cultures grown with histidine. The signal reappeared when 4T12 was propagated in medium lacking histidine. Histidine induced change in nuclear ratio in 4T12 was further confirmed by three tests: (i) inoculum test using conidia, (ii) hyphal tip analysis, and (iii) genetic test using colour markers.
Nuclear ratio and enzyme activity. Because in 4T12 changes in nuclear ratio could be manipulated, this transformant was used to investigate whether the proportion of his-3+t nuclei is correlated with the levels of encoded enzyme, histidinol dehydrogenase. Surprisingly, the specific activity of histidinol dehydrogenase was the same regardless of the percentage of his-3+t nuclei. This observation suggested that the physiological demand of a metabolite may be satisfied with only a few nuclei carrying the relevant gene. Or in other words the majority of nuclei in the coenocytic mycelium may, perhaps, not be active simultaneously.
Silencing of transforming DNA in nuclei. Two experiments were done to test the possibility that in a majority of nuclei, the transforming DNA is selectively silenced by methylation of cytosine: (1) Southern analysis of chromosomal DNA digested with isoschizomers, and (2) Reactivation by growth of transformants in presence of 5-azacytidine, an inhibitor of methylation. The results suggested that a majority of transformed nuclei may, perhaps, be inactive. The results of Northern analysis suggested that the amount of his-3+t transcript was correlated (but 5-azacytidine experiment
indicated that only few his-3+t nuclei may be active) with the proportion of his-3+t nuclei, but not histidinol dehydrogenase activity. The above results suggested that expression of his-3+t gene was controlled both at the levels of transcription and posttranscription.
Nuclear selection. To study competition between nuclei containing mutant (his-3) nuclei and prototrophic nuclei containing his-3+ gene at its normal chromosomal location or at the ectopic location, heterokaryons were synthesized using strains in which the nuclear types had been marked by non-allelic genetic colour markers, al-1 and al-2. The results suggested that in heteronuclear mixture, the replication rate of the transformed nuclei is affected as compared to the nuclei having the gene in normal chromosomal location.
Major contributions. This study generated (his-3 + his-3+) heterokaryons by transformation. The behaviour of transformants differed in some respects both in the vegetative and sexual phases. It was demonstrated that nuclear ratio could be experimentally altered. However, there was no correlation between nuclear ratio and enzyme activity. The observations imply asynchronous division rate among nuclei and raise the possibility that not all nuclei in the coenocytic mycelium are active simultaneously.
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Allosteric Regulation of the First Enzyme in Histidine BiosynthesisLivingstone, Emma Kathrine January 2015 (has links)
The ATP-PRTase enzyme catalyses the first committed step of histidine biosynthesis in archaea, bacteria, fungi and plants.1 As the catalyst of an energetically expensive pathway, ATP-PRTase is subject to a sophisticated, multilevel regulatory system.2 There are two families of this enzyme, the long form (HisGL) and the short form (HisGS) that differ in their molecular architecture. A single HisGL chain comprises three domains. Domains I and II house the active site of HisGL while domain III, a regulatory domain, forms the binding site for histidine as an allosteric inhibitor. The long form ATP-PRTase adopts a homo-hexameric quaternary structure.3,4 HisGS comprises a similar catalytic core to HisGL but is devoid of the regulatory domain and associates with a second protein, HisZ, to form a hetero-octameric assembly.5
This thesis explores the allosteric regulation of the short form ATP-PRTase, as well as the functional and evolutionary relationship between the two families. New insight into the mode allosteric inhibition of the short form ATP-PRTase from Lactococcus lactis is reported in chapter two. A conformational change upon histidine binding was revealed by small angle X-ray scattering, illuminating a potential mechanism for the allosteric inhibition of the enzyme. Additionally, characterisation of histidine binding to HisZ by isothermal titration calorimetry, in the presence and absence of HisGS, provided evidence toward the location of the functional allosteric binding site within the HisZ subunit.
Chapter three details the extensive effort towards the purification of the short form ATP-PRTase from Neisseria menigitidis, the causative agent of bacterial meningitis. This enzyme is of particular interest as a potential target for novel, potent inhibitors to combat this disease. The attempts to purify the long form ATP-PRTase from E. coli, in order to clarify earlier research on the functional multimeric state of the enzyme, are also discussed.
Chapter four reports the investigation of a third ATP-PRTase sequence architecture, in which hisZ and hisGS comprise a single open reading frame, forming a putative fusion enzyme. The engineering of two covalent linkers between HisZ and HisGS from L. lactis and the transfer of the regulatory domain from HisGL to HisGS, is also discussed, in an attempt to delineate the evolutionary pathway of the ATP-PRTase enzymes. Finally, the in vivo activity of each functional and putative ATP-PRTase was assessed by E. coli BW25113∆hisG complementation assays.
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The Role of Histidine-rich Glycoprotein in Angiogenesis and Tumor GrowthThulin, Åsa January 2009 (has links)
Histidine-rich glycoprotein (HRG) is a heparin-binding plasma protein modulating immune, hemostatic and vascular functions. I have studied the antiangiogenic functions of HRG in vitro and in vivo in order to understand the molecular mechanisms of action of HRG as an angiogenesis inhibitor. Angiogenesis is the formation of new blood vessels from the pre-existing vasculature. It is a central rate-limiting step of tumor development and thus a possible target for cancer therapeutics. Previous studies have shown that HRG has antiangiogenic functions in vivo and that the antiangiogenic effects are mediated via the proteolytically released His/Pro-rich domain of HRG. In this thesis we demonstrate that HRG can inhibit endothelial cell migration by interfering with focal adhesion and cytoskeletal turnover. Moreover we have identified the minimal active domain of HRG, a 35 amino acid peptide derived from the histidine- and proline-rich domain of HRG. Analyzing human tumor tissue samples, we have found that a His/Pro-rich fragment of HRG is bound to the vasculature from cancer patients but not to the vasculature from healthy individuals. The fragment is found in association with platelets, and we show that activated platelets can induce a functional microenvironment for the His/Pro-rich fragment. Cancer patients often display an increased coagulation and our data describe a new mechanism to confer specificity of an angiogenesis inhibitor for situations with enhanced platelet activation, as in the tumor. We have further studied the role of HRG in tumor growth by crossing HRG-deficient mice with a transgenic mouse model of pancreatic insulinoma. We show that mice lacking HRG display an elevated “angiogenic switch” and that the total tumor volume is larger in these mice than in wild type mice. HRG is also involved in regulation of platelet function and platelets can stimulate angiogenesis in various ways. We have depleted mice of platelets to study the possible connection between the function of HRG in angiogenesis and platelet regulation. Our data suggest an involvement of platelets in the antiangiogenic activities of HRG.
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Characterization of histidine-tagged NaChBac ion channelsKhatchadourian, Rafael Aharon. January 2008 (has links)
Imaging tools in cellular and molecular biology have long relied on organic fluorophores to observe microorganisms or various cell constituents. The advent of semiconductor nanoparticles known as quantum dots (QDs) has offered the possibility to use this new class of fluorescent probes with very advantageous optical properties in cell biology. The imaging of transmembrane potential and ionic currents is of significant importance for monitoring the activity of the cell. It remains possible with relatively complicated instruments and methods such as patch clamping. A complementary approach to view the dynamics of ion channels with modern and efficient fluorophores is therefore of great interest to the field of biology in general. / We developed a construct based on the FRET signal between QDs and organic fluorescent dyes to monitor the conformational changes of voltage gated sodium channels. The amino acid histidine was used as a "landing platform" for QDs and the bacterial sodium channel NaChBac was chosen for testing. This study focused on the preliminary steps of the project and aimed to characterize the electrophysiological behavior of the histidine-tagged channel. The whole-cell configuration of patch clamping was the tool we used to understand the differences between the wild-type and the histidine-tagged variants of the channels. We also explore the possibility to land QDs on the histidine tag.
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Structural and biochemical analysis of HutD from Pseudomonas fluorescens SBW25 : a thesis submitted in fulfilment of the requirements for the degree of Master of Science in Molecular Biosciences at Massey University, Auckland, New ZealandLiu, Yunhao January 2009 (has links)
Pseudomonas fluorescens SBW25 is a gram-negative soil bacterium capable of growing on histidine as the sole source of carbon and nitrogen. Expression of histidine utilization (hut) genes is controlled by the HutC repressor with urocanate, the first intermediate of the histidine degradation pathway, as the direct inducer. Recent genome sequencing of P. fluorescens SBW25 revealed the presence of hutD in the hut locus, which encodes a highly conserved hypothetical protein. Previous genetic analysis showed that hutD is involved in hut regulation, in such a way that it prevents overproduction of the hut enzymes. Deletion of hutD resulted in a slow growth phenotype in minimal medium with histidine as the sole carbon and nitrogen source. While the genetic evidence supporting a role of hutD in hut regulation is strong, nothing is known of the mechanism of HutD action. Here I have cloned and expressed the P. fluorescens SBW25 hutD in E. coli. Purified HutD was subjected to chemical and structural analysis. Analytic size-exclusion chromatography indicated that HutD forms a dimer in the elution buffer. The crystal structure of HutD was solved at 1.80 Å (R = 19.3% and Rfree = 22.3%) by using molecular replacement based on HutD from P. aeruginosa PAO1. P. fluorescens SBW25 HutD has two molecules in an asymmetric unit and each monomer consists of one subdomain and two ß-barrel domains. Comparative structural analysis revealed a conserved binding pocket. The interaction of formate with a highly conserved residue Arg61 via salt-bridges in the pocket suggests HutD binds to small molecules with carboxylic group(s) such as histidine, urocanate or formyl-glutamate. The hypothesis that HutD functions via binding to urocanate, the hut inducer, was tested. Experiments using a thermal shift assay and isothermal titration calorimetry (ITC) analysis suggested that HutD binds to urocanate but not to histidine. However, the signal of HutD-urocanate binding was very weak and detected only at high urocanate concentration (53.23 mM), which is not physiologically relevant. The current data thus does not support the hypothesis of HutD-urocanate binding in vivo. Although the HutD-urocanate binding was not confirmed, this work has laid a solid foundation for further testing of the many alternative hypotheses regarding HutD function.
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