O uso da nisina como conservante natural tem aumentado cada vez mais, assim como os estudos para aprimorar o uso desta. Este trabalho visa o aumento da eficiência da nisina na inibição de microorganismos, como a Listeria, através da encapsulação da mesma na forma de nanolipossomas com incorporação de polissacarídeos. Foram testados nanolipossomas de nisina e nanolipossomas de nisina com incorporação de quitosana e incorporação de sulfato de condroitina. As análises mostraram que estes lipossomas tiveram a mesma eficiência que a nisina livre na inibição da L. monocytogenes 4b* e da Listeria sp.* e uma maior eficiência na inibição da L. monocytogenes 4b ATCC 6477. As análises de potencial zeta indicaram que os lipossomas de nisina com e sem incorporação de polissacarídeos possuem carga em torno de -20mV e estes apresentaram estruturas esféricas e não esféricas quando observados através de TEM. Apesar dos nanolipossomas de nisina com incorporação de quitosana apresentarem uma eficiência de encapsulaçãos mais baixa, em torno de 10%, estes tiveram maior estabilidade e também maior eficiência na inibição da Listeria monocytogenes ATCC 7644, quando comparados aos nanolipossomas de nisina com e sem incorporação de sulfato de condroitina, no tempo final de 24h a redução obtida foi de 2 log comparando-se com os demais tratamentos e de 7 log comparando-se aos controles. / The use of nisin, as a natural preservative, has increased, as well as studies to enhance the use of that. The aim of this study is to increase the efficiency of nisin in inhibition of microorganisms such as Listeria, by encapsulating as nanolipossomes incorporated with polysaccharides. Nisin nanoliposomes and nisin nanoliposomes with incorporation of chitosan and chondroitin sulfate were analysed. The analyzes showed that these liposomes had the same efficiency as free nisin in inhibiting L. monocytogenes 4b and Listeria sp., both isolated from bovine carcass, and greater efficiency in inhibiting L. monocytogenes 4b ATCC 6477. The zeta potential analyzes indicated that nisin liposomes with and without incorporation of polysaccharides have a load around -20mV and these showed spherical and nonspherical structures when observed through TEM. Despite of the efficience of nisin nanoliposomes incorporating chitosan present lower values of encapsulation efficiency, around 10%, they were more stable and also more efficient in inhibiting Listeria monocytogenes ATCC 7644 compared to nisin nanoliposomes with and without incorporation of chondroitin sulfate. In the final time of 24 hours the reduction obtained was 2 log compared with the other treatments and 7 log comparing to the controls.

O uso da nisina como conservante natural tem aumentado cada vez mais, assim como os estudos para aprimorar o uso desta. Este trabalho visa o aumento da eficiência da nisina na inibição de microorganismos, como a Listeria, através da encapsulação da mesma na forma de nanolipossomas com incorporação de polissacarídeos. Foram testados nanolipossomas de nisina e nanolipossomas de nisina com incorporação de quitosana e incorporação de sulfato de condroitina. As análises mostraram que estes lipossomas tiveram a mesma eficiência que a nisina livre na inibição da L. monocytogenes 4b* e da Listeria sp.* e uma maior eficiência na inibição da L. monocytogenes 4b ATCC 6477. As análises de potencial zeta indicaram que os lipossomas de nisina com e sem incorporação de polissacarídeos possuem carga em torno de -20mV e estes apresentaram estruturas esféricas e não esféricas quando observados através de TEM. Apesar dos nanolipossomas de nisina com incorporação de quitosana apresentarem uma eficiência de encapsulaçãos mais baixa, em torno de 10%, estes tiveram maior estabilidade e também maior eficiência na inibição da Listeria monocytogenes ATCC 7644, quando comparados aos nanolipossomas de nisina com e sem incorporação de sulfato de condroitina, no tempo final de 24h a redução obtida foi de 2 log comparando-se com os demais tratamentos e de 7 log comparando-se aos controles. / The use of nisin, as a natural preservative, has increased, as well as studies to enhance the use of that. The aim of this study is to increase the efficiency of nisin in inhibition of microorganisms such as Listeria, by encapsulating as nanolipossomes incorporated with polysaccharides. Nisin nanoliposomes and nisin nanoliposomes with incorporation of chitosan and chondroitin sulfate were analysed. The analyzes showed that these liposomes had the same efficiency as free nisin in inhibiting L. monocytogenes 4b and Listeria sp., both isolated from bovine carcass, and greater efficiency in inhibiting L. monocytogenes 4b ATCC 6477. The zeta potential analyzes indicated that nisin liposomes with and without incorporation of polysaccharides have a load around -20mV and these showed spherical and nonspherical structures when observed through TEM. Despite of the efficience of nisin nanoliposomes incorporating chitosan present lower values of encapsulation efficiency, around 10%, they were more stable and also more efficient in inhibiting Listeria monocytogenes ATCC 7644 compared to nisin nanoliposomes with and without incorporation of chondroitin sulfate. In the final time of 24 hours the reduction obtained was 2 log compared with the other treatments and 7 log comparing to the controls.

Injury to the central nervous system (CNS) results in the formation of a highly inhibitory glial scar consisting mainly of chondroitin sulfate proteoglycans (CSPGs). CSPGs are comprised of a protein core with covalently attached chondroitin sulfate glycosaminoglycan (CS-GAG) side chains. CSPGs and CS-GAGs have been implicated in the regenerative failure of the CNS, though the mechanism underlying inhibition is unclear. Sulfation affects both the physical and chemical characteristics of CS-GAGs and, therefore, it has been hypothesized that certain sulfation patterns are more inhibitory than others. To investigate this hypothesis, specific chondroitin sulfate sulfotransferases (CSSTs), the enzymes responsible for CS-GAG sulfation, were knocked down in vitro using siRNA. C4ST-1, C4ST-2, and C46ST were chosen as targets for gene knockdown in this study based on their expression in neural tissue and the extent of inhibition caused by their respective CS-GAG. It was hypothesized that transfection of primary rat astrocytes with siRNAs designed to prevent the expression of C4ST-1, C4ST-2, and C46ST would decrease specific sulfation patterns of CSPGs, resulting in improved neurite extension in a neurite guidance assay. Through optimization of siRNA dose, astrocyte viability was maintained while successfully knocking down mRNA levels of C4ST-1, C4ST-2, and C46ST and significantly reducing total levels of secreted CS-GAGs. However, no increase in the incidence of neurite extension was observed using conditioned media collected from siRNA transfected astrocytes compared to non-transfected controls. These data suggest that sulfation does not contribute to CSPG-mediated neurite inhibition, though further investigation is necessary to confirm these findings. Significantly, this work has established a paradigm for investigating the role of CSPG sulfation patterns in CNS regeneration.

In all living organisms, biological activities such as proper functioning and co-ordination of different organs will depend on different cells and molecular interactions. In some organisms the loss of functional organs or damage of organs can be lethal, whereas in others a special process called regeneration can retrieve lost organs. The molecular details of regeneration are still not completely understood in many organisms. Echinoderms are close to vertebrates in the evolutionary tree and are well known for their amazing regeneration capacity. So we chose to investigate the molecular processes of regeneration mechanism with an interest towards our favorite groups of molecules, glycosaminoglycans (GAGs). GAGs are linear polysaccharides, expressed on all cell surfaces and extracellular space and are also known to be involved in many cellular activities. We aimed to characterize the GAGs present in Echinodermata species Amphiura filiformis and investigated their role during arm regeneration. In Paper I we characterized the structure and function of GAGs from A. filiformis and identified that A. filiformis contains CS/DS type of GAGs, but no HS. The sulfation degree of these CS/DS is close to the one of heparin, i.e. they are highly sulfated. These chains are able to bind FGF-2 growth factor and induce FGF-2 mediated cell signaling. In Paper II we further characterized these GAGs for their localization and for their role in arm regeneration in A. filiformis. Immuno- and histochemical stainings on arm sections revealed that CS/DS GAGs are localized around the podia, surrounding the water vascular system, and around the muscle tissues. Inhibition of sulfated GAG biosynthesis by chlorate treatment affected the regeneration efficiency of the arms, which may be an indication of the importance of CS/DS structures in A. filiformis arm regeneration. We also characterized some bacterial sulfatases in Paper III and a lyase in Paper IV from human and canine gut symbiotic bacteria. Here we sought to find the substrate specificity and optimal conditions for these enzymes’ activities. Our findings suggest that these polysaccharide lyase and sulfatases can be used as potential tools to characterize different GAG structures and their application could further add knowledge on diseases mechanisms related to host pathogen interactions.

Chondroitin sulfate proteoglycans (CSPGs) are found in specialized brain extracellular matrix structures termed perineuronal nets (PNNs). The chondroitin sulfate chains of these CSPGs are thought to have a strong effect on neuroplasticity, along with development, injury, and diseased states of the brain. Wisteria floribunda agglutinin (WFA) is a plant lectin used to identify PNN via staining; the pattern of this staining is changed upon schizophrenia. As such, one powerful method of probing the identity of the CS chains of PNNs and addressing what changes in CS identity occur during schizophrenia is to characterize the features of the CS which bind to the lectin. Methods for characterization of WFA-CS binding and their biological relevance were developed and evaluated. Commercially available CS was used to probe the binding affinity of the agglutinin to various regions of CS via hemagglutination inhibition assays and affinity gradient elution of CS bound to WFA. The size, sulfation extent, and fragment location in the CS chain from these eluates were determined using HILIC-LC-MS. As commercial sources can be used to elucidate the binding specificity of WFA, but not the actual relevant binding partner of WFA within the brain, PNN CS extractions were performed with a modified method aimed at reducing the timescale at which PNN CS can be obtained so as to allow similar experimentation on CS directly from PNN. The results pave the way for further determination of WFA-CS binding.

Tissue inhibitor of metalloproteinase-3 (TIMP-3) is a protein with multiple functions that include regulating the turnover of the extracellular matrix (ECM) by inhibiting members of the metzincin family. Extracellular levels of soluble TIMP-3 are low, reflecting its binding to components of the ECM including sulfated glycosaminoglycans (SGAGs) and its endocytosis by low density lipoprotein receptor-related protein 1. Because TIMP-3 inhibits ECM-degrading enzymes, the ability of SGAG mimetics to elevate extracellular concentrations of TIMP3 is of interest for osteoarthritis treatment. However, previous studies of such interactions have utilized immobilized forms of the protein or ligands. Here we have quantified the thermodynamics of the interactions of the inhibitory domain of TIMP-3 with chondroitin sulfate (CS), pentosan polysulfate (PPS) and suramin in solution using isothermal titration calorimetry. All three interactions are driven by a (favorable) negative enthalpy ychange combined with an unfavorable decrease in entropy. The heat capacity change (ΔCp) for the interaction of N-TIMP-3 with CS, PPS, or suramin is essentially zero, indicating an insignificant contribution from the hydrophobic effect. Based on the effects of ionic strength on the interaction of N-TIMP-3 with suramin, their interaction appears to be driven by electrostatic interactions. Modeling supports the view that the negatively charged sulfates of CS, PPS, and suramin interact with a cationic region on N-TIMP-3 that includes Lys -26, -27, -30, and -possibly 76 on the opposite face of TIMP-3 from its reactive site for metalloproteases. / Includes bibliography. / Dissertation (Ph.D.)--Florida Atlantic University, 2019. / FAU Electronic Theses and Dissertations Collection

名古屋大学博士学位論文 学位の種類 : 博士(医学)(課程) 学位授与年月日:平成6年4月5日 西田佳弘氏の博士論文として提出された

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function.

Chondroitin sulphate proteoglycans (CSPGs) from the glial scar inhibit axonal regeneration following spinal cord injury. CSPG expression can be induced by transforming growth factor β (TGFβ), which suggests that inhibition of TGFβ may reduce CSPG levels. Astrocytes were treated with cyclic AMP (cAMP), which reduced TGFβ signaling protein Smad2 in astrocytes. However, cAMP-treated astrocytes showed strong neurocan expression following TGFβ treatment, which suggests that TGFβ may mediate CSPG expression through non-Smad pathways. Smad2 or Smad4 were knocked down in astrocytes using siRNA and TGFβ-induced neurocan, brevican and aggrecan expression were still observed, indicating that Smad signaling is not required for CSPG expression. Administration of a PI3K/Akt inhibitor produced significant reductions in neurocan, brevican and aggrecan expression in astrocytes, which suggests that PI3K/Akt pathway mediates CSPG expression. Erk1/2 inhibitor treatment did not reduce CSPG expression significantly. Targeting non-Smad signaling pathways may therefore be effective strategies to reduce CSPG expression following injury.

The proteoglycan CD44 is a widely expressed cell surface receptor for the extracellular matrix glycosaminoglycan hyaluronan, and is involved in processes ranging from metastasis to wound healing. In the immune system, leukocyte activation induces hyaluronan binding through changes in CD44 post-translational modification, but these changes have not been well characterized. Here I identify chondroitin sulfate addition to CD44 as a negative regulator of hyaluronan binding. Chondroitin sulfate addition was analyzed by sulfate incorporation and Western blotting and determined to occur at serine 180 in human CD44 using site-directed mutagenesis. Mutation of serine 180 increased hyaluronan binding by both a CD44-immunoglobulin fusion protein expressed in HEK293 cells, and full-length CD44 expressed in murine L fibroblast cells. In bone marrow-derived macrophages, hyaluronan binding induced by the inflammatory cytokines tumor necrosis factor-α and interferon-γ corresponded with reduced chondroitin sulfate addition to CD44. Retroviral infection of CD44⁻/⁻ macrophages with mouse CD44 containing a mutation at serine 183, equivalent to serine 180 in human CD44, resulted in hyaluronan binding that was constitutively high and no longer enhanced by stimulation. These results demonstrate that hyaluronan binding by CD44 is regulated by chondroitin sulfate addition in macrophages. A functional consequence of altered chondroitin sulfate addition and increased hyaluronan binding was observed in Jurkat T cells, which became more susceptible to activation-induced cell death when transfected with mutant CD44. The extent of cell death was dependent upon both the hyaluronan binding ability of CD44 and the size of hyaluronan itself, with high molecular mass hyaluronan having a greater effect than intermediate or low molecular mass hyaluronan. The addition of hyaluronan to pre-activated Jurkat T cells induced rapid cell death independently of Fas and caspase activation, identifying a unique Fas-independent mechanism for inducing cell death in activated cells. Results were comparable in splenic T cells, where high hyaluronan binding correlated with increased phosphatidylserine exposure, and hyaluronan-dependent cell death occurred in a population of restimulated cells in the absence of Fas-dependent cell death. Together these results reveal a novel mechanism for regulating hyaluronan binding and demonstrate that altered chondroitin sulfate addition can affect CD44 function.