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Muropeptidase (MurO) as Surface Protein Anchor for Anti HIV-1 2F5 Epitope on Lactobacillus plantarumHussain, Rizwana January 2014 (has links)
Human immunodeficiency virus type 1 (HIV-1) is a major public health concern because it cannot yet be prevented by vaccination. Mucosal surfaces are the primary sites for the HIV-1 transmission. Vaccine capable of protecting HIV-1 depends on the induction of long term mucosal immune responses. In the infected individuals, anti HIV-1 epitope can generate neutralizing antibodies and have protective effects. Vaccine which can induce local mucosal immunity may prevent HIV-1 replication within local tissues prior to systemic dissemination. The 2F5 human monoclonal antibody (MAb) has HIV-1 neutralizing activity. A conserved linear sequence ELDKWA which is present within the envelope glycoprotein of HIV virus is an epitope of 2F5 MAb. Expression of anti HIV-1 ELDKWA epitope on the cell surface of L. plantarum via anchor protein can be a strategy to develop HIV-1 vaccines. Lactobacillus plantarum are probiotics and have cell surface displaying ability. The aim of this study was to express anti HIV-1 2F5 epitope ELDKWA on the L. plantarum NC8 cell surface using muropeptidase (MurO) of L. plantarum CCUG 9289. The expression of recombinant protein MurO. 2F5 in L. plantarum NC8 was determined by Western blot following cloning techniques. BLASTP analysis showed that MurO has two putative lysine motif (LysM) domains that can bind to peptidoglycan. Our results suggest that MurO of L. plantarum can be used as an anchor protein for anti HIV-1 ELDKWA epitope expression in L. plantarum. This implies that using L. plantarum together with anchor protein MurO could effectively be used as a vaccine delivery system against HIV-1 infection.
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Lysine Analogue Use and Thromboembolic Risks: An Evidence Based AnalysisMontroy, Joshua January 2018 (has links)
Allogenic blood transfusions, although potentially life-saving, are associated with increased risk of infection, fluid overload, thrombosis, and death. Minimizing exposure to blood products is important for patients and the healthcare system. Antifibrinolytic lysine analogues are effective antihaemorrhagic agents used for the reduction of blood loss and subsequent need for transfusion. However, the pharmacologic mechanism of decreased clot breakdown would suggest the medications should increase the risk of venous thromboembolic adverse events. Trials of lysine analogue administration are often underpowered to detect the effect of these medications on thrombotic events. As lysine analogue use increases for blood loss reduction, there is an important need for research dedicated to the safety of lysine analogues, especially in patients who are at highest risk of venous thromboembolism.
Through systematic review and meta-analyses, we investigated the use of lysine analogues in cancer patients, where VTE is particularly prevalent. We identified that only a small number of trials have been performed in cancer patients. Among available data, similar reduction of transfusion was observed in cancer patients treated with lysine analogues compared to non-cancer patients. However, we also found that existing data was grossly underpowered to determine the effect of lysine analogues on risk of VTE (Peto odds ratio (OR) 0.60; 95% CI 0.28-1.30).
By administering lysine analogues topically, as opposed to intravenously, systemic absorption of the drug may be limited, and the occurrence of unwanted side-effects may be minimized. We also reviewed the published literature to determine if there was sufficient evidence to support topical application of tranexamic acid. Topically applied tranexamic acid effectively reduces both transfusion risk and blood loss and no increased risk of VTE events was observed (pooled OR=0.78, 95% CI 0.47 to 1.29). However, none of these studies included cancer patients and the vast majority of the trials were in orthopedic surgery.
Lastly, we sought to determine the extent to which lysine analogues are currently used at a large tertiary care academic institution. In addition, we explored which factors influenced lysine analogue use, and areas of informational or study need. Surgeons reported low lysine analogue use, and the timing of administration varied considerably. Many surgeons (66%) believed a clinical trial was needed to demonstrate the efficacy of lysine analogues in their respective surgical field, and 59% felt a trial was needed to demonstrate that the medication was safe in their patient population.
We confirmed that there are only a few studies evaluating the effect of lysine analogues in cancer patients and that many surgeons are concerned about the safety profile of these medications. Surgeons may feel more comfortable administering these agents topically as opposed to intravenously, and while this may be a safer option, there has been limited evaluation of this approach outside of orthopedic procedures.
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Expanding the network of enzymes affecting methylation at H3K4 (histone 3 lysine 4) during Caenorhabditis elegans embryogenesisWilkins, Elizabeth January 2016 (has links)
Post translational modifications (PTMs) of histone tails are an important determinant of chromatin structure, and can act as key regulators of DNA-dependent processes. Methylation of histone 3 at lysine 4 (H3K4) is one of the most widely studied PTMs because of its correlation with transcription. Three methylation states exist at H3K4: mono-, di, and tri-methylation (H3K4me1, -me2, and -me3, respectively). Each methylation state occupies a distinct genomic position, supporting the view that the extent of methylation at H3K4 has a functional significance. However, the exact biological function of these three marks are not well understood. H3K4 methylation is written by SET domain-containing enzymes that function within SET/COMPASS/MLL complexes. Our lab has previously identified the SET-16 enzyme as writing H3K4me3 in C. elegans. The other well-characterised H3K4-specific methyl transferases in the worm is SET-2, an enzymes responsible for bulk H3K4me2/me3 levels. Using targeted RNAi screens, we have characterised the full landscape of SET domain enzymes affecting all three methylation states at H3K4 during embryogenesis in C. elegans (Chapter 3). Unexpectedly, many previously uncharacterised enzymes were identified as preferentially affecting each of the methylation states, including SET-19 that can deposit all three marks, and several candidates that preferentially affect H3K4me1: SET-30, SET-27, MES-2, and MES-4. During the project, Greer et al. 2014 independently identified two enzymes with activities targeting H3K4, SET-17 and SET-30, which were also candidates from our RNAi screens. With a focus on enzymes acting on H3K4me1, we demonstrate that H3K4me1 candidates can show different patterns of temporal regulation and also have roles in regulating soma versus germline cell-fate decisions (Chapter 4). Finally, we demonstrate a novel role for MES-2 (a methyltransferase enzyme with a highly conserved role in depositing repressive H3K27 methylation) in acting alongside the SPR-5 H3K4me2 demethylase to regulate levels of H3K4me1 during embryogenesis (Chapter 5).
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Effect of RNAi down-regulation of three lysine-deficient kafirins on the seed lysine content of sorghum [Sorghum bicolor (L.) Moench]Grootboom, Andile W. 23 October 2010 (has links)
Sorghum (Sorghum bicolor L. Moench) ranks fifth worldwide in production among cereals. It is a major staple food for millions in Africa and Asia, and a major livestock feed grain in developed countries. However, the sorghum grain is poor in lysine content, limiting its value as food and feed. In this study, I hypothesize that reduction of some of the major storage proteins that are inherently poor in lysine through in vitro manipulation will result in the enhanced expression of proteins with a better lysine profile and, thus, increased overall grain lysine content. Sorghum genotypes were screened for in vitro amenability and a sorghum genotype-tissue culture medium combination that yielded the highest somatic embryo callus formation and regeneration potential, was identified. This resulted in the establishment of a sorghum biolistic transformation method with a transformation efficiency of 3.36%, the highest reported to date. Using genetic engineering tools, the enhancement of the nutritional quality of grain sorghum was achieved by increasing the seed lysine content. An RNAi co-suppression strategy was employed and resulted in 45.23 and 77.55% increase in whole seed and endosperm lysine increase, respectively. The co-suppression RNAi constructs targeted the endosperm specific suppression of three lysine-poor storage proteins, namely ä-kaf-2, ã-kaf-1 and -2, and an enzyme that catalyzes seed lysine degradation, lysine keto-gluterate reductase (LKR). Seven independent transgenic events displayed successful transgene integration for both the selectable marker gene and the target constructs. However, the Southern blot hybridization analysis revealed two transgenic events that displayed transgene re-arrangement at the 5’promoter end, thus resulting in a lack of suppression of target proteins. Variations in target proteins co-suppression was observed with Western blot analysis and RT-PCR for both the target kafirins and LKR suppression, and no lysine improvement was observed where no kafirin suppression occurred. The transgenic co-suppression of the target kafirins resulted in the endosperm structural change from a hard, corneous endosperm to a soft, floury endosperm, consistent with ã-zein suppression in the Opaque-2 maize mutant. / Thesis (PhD)--University of Pretoria, 2010. / Plant Science / unrestricted
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Identification of Non-histone Acetylation Targets in Saccharomyces cerevisiaePourhanifeh-Lemeri, Roghayeh 06 June 2012 (has links)
Lysine acetylation is a conserved post-translational modification (PTM) which was traditionally believed to be limited to histones and the regulation of gene expression. However, recent proteomic studies have identified lysine acetylation on proteins implicated in virtually all cellular processes indicating that this PTM plays a global regulatory role. Indeed, in humans, aberrance of lysine acetyltransferase (KAT) activity is associated with various pathogenesis. To date, over 2500 human proteins are known to be acetylated in vivo, but very few acetylations have been linked to specific KATs. Hence, to understand the biological relevance of KATs and acetylation in human pathology, it is important to learn about the mechanism regulating KAT activity and the identity of their in vivo targets. This is a complex task and will require the use of model organisms and system biology approaches. The work presented here explores the significance of self-acetylation in regulating KAT function by focusing on the highly NuA4 lysine acetyltransferase in the model organism Saccharomyces cerevisiae or budding yeast. Using genetics and biochemical assays I have identified NuA4 subunit Epl1 as a novel in vivo NuA4 substrate. I have also shown that Epl1 acetylation regulates NuA4 function at elevated temperatures. In an attempt to identify new biological processes regulated by yeast KATs and putative novel substrates, I have also performed a genome-wide synthetic dosage lethality screen with six non-essential yeast KATs; Hat1, Rtt109, Hpa2, Sas3, Sas2, and Elp3. My screen identified largely distinct sets of genetic interactions for each KAT suggesting that each KAT has specific cellular functions. Together, this study demonstrates the importance of auto-acetylation in regulating KAT function and the diversity of cellular processes impacted by KAT activity in vivo.
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Antibody-free affinity enrichment for global methyllysine discoveryDewar, Charlotte 20 December 2019 (has links)
Lysine methylation is a post-translational modification that regulates a large array of functionally diverse processes that are vital for cellular function. The role of methylation is best characterized on histone proteins due to their high concentration in the cell, but alongside histone modifications, lower abundance non-histone methylation is emerging as a prevalent and functionally diverse regulator of cellular processes. The direct biological impact of non-histone lysine methylation is less well understood because they are difficult to detect. The dynamic concentration range of the proteome masks their signal during proteomic analysis which impedes the detection of these low abundance methylated proteins. Increasing the concentration of proteins bearing methylation is required for improved discovery. This requires enriching the post-translational modification with a capturing reagent prior to analysis.
This thesis details an optimized method for using the supramolecular host p-sulfonatocalix[4]arene as a stationary phase methyllysine enrichment reagent for real-life cell-extracted proteins. Prior to the optimizations described in this thesis, cell-derived peptide extracts were not retained within an early generation upper-rim modified calixarene column. But with the new protocols detailed in this thesis, proteins extracted from both cultured prostate cancer cells and industrially sourced brewer’s yeast were successfully retained by a lower-rim modified calixarene column. Thousands of methylated proteins with diverse functions and cellular localization were discovered using this method. Detection of low abundance methylated proteins will aid our discovery of all cellular methylation marks, which in turn, will help delineate their biological functions. / Graduate / 2020-11-30
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Characterization of T1rho sensitivity to metabolite and temperature changesOwusu, Nana 01 July 2015 (has links)
Spin-lattice relaxation in the rotating frame (T1ρ) is a relaxation parameter measured in nuclear magnetic resonance studies. This parameter has been found to be sensitive to chemical exchange processes occurring in diseased tissue associated with abnormal metabolism when measured in magnetic resonance imaging (MRI). Metabolic changes in tissue affected by abnormal metabolism can be quantified with good spatial and temporal resolution using T1ρ, better than a similar method of assessment known as CEST and current methods such as spectroscopic (1H- and 31P-MRS) and nuclear medicine (PET) methods used in clinical settings. Though T1ρ has these advantages, there is no consensus as to which metabolic changes T1ρ is most sensitive. The metabolic changes may be pH related, or due to changes in concentration of metabolites like glucose and glycogen. This work is tries to identify which metabolite evokes the greatest change in T1ρ by studying the response of three spin relaxation measures (T1ρ, T2 and T1) at different temperatures. It was found that T1ρ is more sensitive to pH changes than glucose and lysine at 3T. Also at body temperature, the pH results showed an exponential decay trend for T1ρ signifying the limited range of sensitivity in the pH range of 6.9 to 7.5. The T2 results can be used to explain this trend.
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Bone phenotype of lysyl oxidase isoform knockout mice & in vitro expression of lysyl oxidase proenzymeAlsofi, Loai A. January 2008 (has links)
Thesis (D.Sc.D.)--Boston University, Goldman School of Dental Medicine, 2008 (Dept. of Periodontology and Oral Biology). / Includes bibliographical references: leaves 140-148. / Lysyl oxidases constitute a family of enzymes responsible for the formation of cross
links in collagen and elastin. These enzymes have also been linked to pathological fibrosis.
The importance of collagen in the structural and mechanical properties of bone led us to
investigate the hypothesis that the absence of one or more of these enzymes could lead to a
significant bone phenotype. This phenotype could resemble osteoporosis or diabetic bone
disease. In addition, we tried to overexpress lysyl oxidase proenzyme in vitro. The ability to
produce enough amounts of lysyl oxidase proenzyme and the ability to process it and activate
it could facilitate the development of drugs that control its activity in pathological fibrosis.
Bones from 12-week old mice (8 males and 8 females) with the compound
genotype LOX+/-, LOXLl -/- were analyzed. 5 males of the genotype LOX+/+, LOXLl-/were
also analyzed. 16 wild type mice (8 males and 8 females) were used as controls. μCT
was used to analyze the trabecular and cortical bone morphology of both left femur and L5
vertebrae (n=5). The femora were subsequently subjected to mechanical testing using the
twist failure in torsion. Right femurs (n=5) were used for histology and histromorphometric
analysis. Tibia and fibula (n=5) were used for cross-link analysis. Two way factor ANOV A
with post-hoc Tukey HSD test was used for statistical analysis. A P value of less than 0.05
was used to declare significance. μCT analysis of the trabecular bone in femur distal ... [TRUNCATED]
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Design, synthesis and evaluation of calix[4]arene based enrichment agents for N-methyl proteomicsShaurya, Alok 06 January 2021 (has links)
Role of N-methylated lysines (K) and arginines (R) was underappreciated for a long time before the turn of the century. With the help of new emerging technologies, their crucial role in chromatin regulation was established and now their mention when discussing gene regulation is almost a given. Despite this, much about how they contribute to the cellular chemistry is still to be discovered. There is a major gap in current knowledge base due to an incomplete list of possible lysine and arginine methylation sites. This is because of their low copy number inside the cell which makes it difficult to detect them. New methylation sites are being added every day.
This thesis aims to provide a solution to this problem by establishing methods that can help detect N-methylated lysines and argnines that are present in really low quantity inside the cell. The work is influenced by a previously established fact that p-sulfonatocalix[4]arene binds methylated lysine over unmethylated ones. We have first attempted to improve this native affinity by decorating the calix[4]arene skeleton with different substituents. To this effect, we have developed methods for regioselective functionalization of calix[4]arene scaffold and then studied their effect on its binding profile against a set of test peptides derived from proteins found in vivo.
We then demonstrate a proof-of-concept enrichment method using selected molecules from our inventory. We use these calix[4]arene based molecules as a stationary phase modifier in a chromatography setup and then show that it can separate peptides based on presence of N-methylated lysines and arginines. We propose that introduction of such a method would improve the visibility of low level N-methylated peptides by removing the bulk of back ground unmethylated analytes and thus improving their signal strength. Finally, we establish the utility of this method by showing that more N-methylated lysines are detected from a real-world proteomics sample prepared using our enrichment method.
This work opens new avenues for use of supramolecular chemistry in proteomics studies. We believe that this thesis is a confident demonstration that host-guest chemistry can help expand the existing knowledge about bimolecular processes found in vivo and must be explored further. / Graduate / 2021-12-23
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Identification of the lysine methyltransferase involved in the methylation of VEGFR-2Ruediger, Danielle 03 July 2018 (has links)
Angiogenesis is the process of new blood vessel growth from preexisting vessels. This process relies on the activity of Vascular Endothelial Growth Factor Receptor-2 (VEGFR-2) and occurs in both normal and pathological tissues. Angiogenesis is often dysregulated in diseases such as cancer and many efforts have been made to treat such diseases by targeting the VEGFR-2 pathway. VEGFR-2 is activated upon ligand binding and subsequent autophosphorylation of tyrosine residues in the kinase domain, which leads to endothelial cell survival, proliferation, and growth – all of which are required for angiogenesis to occur.
It was previously demonstrated that methylation of VEGFR-2 at Lys1041 enhanced its tyrosine autophosphorylation and is required for VEGFR-2 mediated angiogenesis in zebrafish and tumor growth in mouse. However, the Lysine Methyltransferase (KMT) involved in the methylation of VEGFR-2 remains unknown. This study aimed to identify the KMT involved in the methylation of VEGFR-2.
We have identified Enhancer of zeste homolog 2 (EZH2) as the KMT likely responsible for catalyzing methylation of K1041 on VEGFR-2. Over-expression of EZH2 was found to increase phosphorylation of Tyr1054, one of the required phosphorylation sites for VEGFR-2 activation, in whole cell lysates and VEGFR-2 purified by immunoprecipitation. The effect of over-expression of EZH2 in the phosphorylation of VEGFR-2 at Tyr1054 was dose-dependent - increasing concentrations of EZH2 resulted in increasing phosphorylation of VEGFR-2 at Tyr1054. Moreover, we determined that EZH2 physically interacts with VEGFR-2 as demonstrated by co-immunoprecipitation in vitro GST-pulldown assays. The C-terminus of EZH2 (amino acids 371-746), physically interacted with VEGFR-2. Taken together, we have identified EZH2 as a candidate KMT involved in the methylation of Lys1041, which increases phosphorylation of VEGFR-2 at Tyr1054. / 2020-07-03T00:00:00Z
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