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Beck, Ane Marie - One of the best experts on this subject based on the ideXlab platform.
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Preparation of Site-Directed Mutants in Human Chitotriosidase
Norwegian University of Life Sciences Ås, 2012Co-Authors: Beck, Ane MarieAbstract:Kitin er et uløselig og lineært polysakkarid som består av N-acetylglukosaminenheter bundet sammen med β-1,4-bindinger. Kitin finnes ikke naturlig i kroppen, men mennesker kan likevel produsere humane kitinaser som degraderer kitin. De to humane kitinasene vi kjenner til i dag er Acidic Mammalian Chitinase (AMCase) og Human Chitotriosidase (HCHT). Både AMCase og HCHT er klassifisert som familie 18 kitinaser. De to humane kitinasene er en del av vårt medfødte immunsystem. HCHT er vist blant annet å motvirke soppinfeksjoner og kan være kilde til nyttige kitooligosakkarider ved transglykosylering. Det er blitt observert produksjon av AMCase og HCHT i sammenheng med mange ulike sykdommer som for eksempel henholdsvis astma og Gauchers sykdom. Inhibering av AMCase har blitt forslått som behandlingsmetode for astma. Dersom HCHT har en positiv effekt på immunforsvaret kan det være ugunstig å inhibere denne kitinasen. For å finne selektive inhibitorer for AMCase og HCHT er det interessant å studere ulikheter mellom bindesetene til disse to enzymene. Målet med denne oppgaven er å utføre seterettet mutagenes på ulike aminosyrer i bindesetet til HCHT. Det skal utføres mutasjoner som endrer aminosyrer i HCHT til aminosyren ved tilsvarende posisjon i AMCase. Mutasjonene som ble utført til dette formålet var Q145R, Y190N, Q188I og S240Y i subsetene +2 og +3. Det ble også utført seterettet mutagenese på to aminosyrer som forventes å være gunstig ved transglykosylering; disse mutasjonene var W218F ved subsete +2 og D138N i TIM-barrel ved subsete -1. Det ble gjennomført vellykkede fremstilling av alle de seks seterettede mutantene. Alle mutantene viste kitinaseaktivitet ved overekspresjon i Pichia pastoris. I tillegg ble AMCase-villtype forsøkt isolert ved ionebyttekromatografi. Ved isolering av AMCase ble det benyttet samme betingelser som for tidligere isolering av HCHT. Det viste seg at ionebytterkromatografi under disse betingelsene ikke ga vellykket isolering av AMCase. Dette kan for eksempel være på grunn av at AMCase har lavere pH optimum enn HCHT.Chitin is an insoluble and linear polysaccharide consisting of N-acetylglucosamine units connected by β-1,4-bonds. Chitin is not produces in the human body, but humans can still produce chitinases that degrades chitin. The two human chitinases known today is Acidic Mammalian Chitinase (AMCase) and Human Chitotriosidase (HCHT). Both AMCase and HCHT are classified as family 18 chitinases. These two human chitinases are a part of our innate immune system. HCHT has been shown to have an anti-fungal effect and can be a source of chito-oligosaccharides by transglycosylation. It has been observed that AMCase and HCHT are produced in the presence of many different Diseases such as asthma and Gauchers Disease, respectively. Inhibition of AMCase has been suggested as treatment for asthma. If HCHT has a positive effect on the immune system it can be unfavorable to inhibit this chitinase. In order to find selective inhibitors of AMCase and HCHT it is interesting to study the differences between the active sites of the two enzymes. The aim of this thesis is to perform site-directed mutagenesis of various amino acids in the active site of HCHT. Amino acids in HCHT are going to be altered to the amino acids at the corresponding position in AMCase. The mutants made for this purpose were Q145R, Y190N, Q188I and S240Y, located by subsites +2 and +3. It was also performed site-directed mutagenesis on two amino acids that are expected to be beneficial for transglycosylation; these mutations were W218F close to subsite +2 and D138N in the TIM-barrel by subsite -1. All the six mutants where successfully produced and all mutants showed chitinase activity by overexpression in Pichia pastoris. In addition, it was attempted to isolate AMCase-wild-type by ion change chromatography. The conditions used for the isolation of AMCase were the same conditions used for previous isolation of HCHT. It turned out that ion change chromatography with these conditions did not give successful isolation of AMCase. This may be because AMCase has a lower pH- optimum than HCHT
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Tillaging av Seterettede Mutanter i Human Chitotriosidase
Norwegian University of Life Sciences Ås, 2012Co-Authors: Beck, Ane MarieAbstract:Kitin er et uløselig og lineært polysakkarid som består av N-acetylglukosaminenheter bundet sammen med β-1,4-bindinger. Kitin finnes ikke naturlig i kroppen, men mennesker kan likevel produsere humane kitinaser som degraderer kitin. De to humane kitinasene vi kjenner til i dag er Acidic Mammalian Chitinase (AMCase) og Human Chitotriosidase (HCHT). Både AMCase og HCHT er klassifisert som familie 18 kitinaser. De to humane kitinasene er en del av vårt medfødte immunsystem. HCHT er vist blant annet å motvirke soppinfeksjoner og kan være kilde til nyttige kitooligosakkarider ved transglykosylering. Det er blitt observert produksjon av AMCase og HCHT i sammenheng med mange ulike sykdommer som for eksempel henholdsvis astma og Gauchers sykdom. Inhibering av AMCase har blitt forslått som behandlingsmetode for astma. Dersom HCHT har en positiv effekt på immunforsvaret kan det være ugunstig å inhibere denne kitinasen. For å finne selektive inhibitorer for AMCase og HCHT er det interessant å studere ulikheter mellom bindesetene til disse to enzymene. Målet med denne oppgaven er å utføre seterettet mutagenes på ulike aminosyrer i bindesetet til HCHT. Det skal utføres mutasjoner som endrer aminosyrer i HCHT til aminosyren ved tilsvarende posisjon i AMCase. Mutasjonene som ble utført til dette formålet var Q145R, Y190N, Q188I og S240Y i subsetene +2 og +3. Det ble også utført seterettet mutagenese på to aminosyrer som forventes å være gunstig ved transglykosylering; disse mutasjonene var W218F ved subsete +2 og D138N i TIM-barrel ved subsete -1. Det ble gjennomført vellykkede fremstilling av alle de seks seterettede mutantene. Alle mutantene viste kitinaseaktivitet ved overekspresjon i Pichia pastoris. I tillegg ble AMCase-villtype forsøkt isolert ved ionebyttekromatografi. Ved isolering av AMCase ble det benyttet samme betingelser som for tidligere isolering av HCHT. Det viste seg at ionebytterkromatografi under disse betingelsene ikke ga vellykket isolering av AMCase. Dette kan for eksempel være på grunn av at AMCase har lavere pH optimum enn HCHT.Chitin is an insoluble and linear polysaccharide consisting of N-acetylglucosamine units connected by β-1,4-bonds. Chitin is not produces in the human body, but humans can still produce chitinases that degrades chitin. The two human chitinases known today is Acidic Mammalian Chitinase (AMCase) and Human Chitotriosidase (HCHT). Both AMCase and HCHT are classified as family 18 chitinases. These two human chitinases are a part of our innate immune system. HCHT has been shown to have an anti-fungal effect and can be a source of chito-oligosaccharides by transglycosylation. It has been observed that AMCase and HCHT are produced in the presence of many different Diseases such as asthma and Gauchers Disease, respectively. Inhibition of AMCase has been suggested as treatment for asthma. If HCHT has a positive effect on the immune system it can be unfavorable to inhibit this chitinase. In order to find selective inhibitors of AMCase and HCHT it is interesting to study the differences between the active sites of the two enzymes. The aim of this thesis is to perform site-directed mutagenesis of various amino acids in the active site of HCHT. Amino acids in HCHT are going to be altered to the amino acids at the corresponding position in AMCase. The mutants made for this purpose were Q145R, Y190N, Q188I and S240Y, located by subsites +2 and +3. It was also performed site-directed mutagenesis on two amino acids that are expected to be beneficial for transglycosylation; these mutations were W218F close to subsite +2 and D138N in the TIM-barrel by subsite -1. All the six mutants where successfully produced and all mutants showed chitinase activity by overexpression in Pichia pastoris. In addition, it was attempted to isolate AMCase-wild-type by ion change chromatography. The conditions used for the isolation of AMCase were the same conditions used for previous isolation of HCHT. It turned out that ion change chromatography with these conditions did not give successful isolation of AMCase. This may be because AMCase has a lower pH- optimum than HCHT
Vesna Zadnik - One of the best experts on this subject based on the ideXlab platform.
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Clinical Study Echocardiographic Assessment of Left Ventricular Function in Type 1 Gaucher’s Disease
2013Co-Authors: Samo Zver, Vesna ZadnikAbstract:Copyright © 2010 Mirta Koˇzelj et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. There is predominate opinion among physicians managing type 1 Gauchers ’ Disease (GD) that cardiac involvement is not an issue in these patients. In order to follow this hypothesis, we prospectively investigated 15 adult imiglucerase-treated type 1 GD patients by echocardiography, Doppler, and tissue Doppler echocardiography. This was a case-controlled study with 18 matched healthy volunteers. The obtained data was correlated with the levels of NT-proBNP (brain natriuretic peptide). None of the GD patients had clinical signs of heart Disease. In 3 of the 15 patients, we observed echocardiographic signs of aortic and mitral valve calcification. The left ventricular systolic function was within normal limits. Compared to the control group, there was no statistically significant difference observed in the most sensitive indices of left ventricular diastolic function, parameter Em (P =.095), and E/Em ratio (P =.097), as demonstrated by tissue Doppler echocardiography. However, there was a positive correlation between the E/Em ratio and NT-proBNP plasma levels (P =.009). In conclusion, the prospective echocardiographic study of type 1 GD patients did not validate any left ventricular dysfunction. But, the E/Em ratio showed a strong statistical correlation with the most sensitive indicators of heart failure, NT-proBNP. Research on larger groups of patients and the usage o
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Echocardiographic Assessment of Left Ventricular Function in Type 1 Gaucher's Disease
Hindawi Limited, 2010Co-Authors: Mirta Koželj, Samo Zver, Vesna ZadnikAbstract:There is predominate opinion among physicians managing type 1 Gauchers' Disease (GD) that cardiac involvement is not an issue in these patients. In order to follow this hypothesis, we prospectively investigated 15 adult imiglucerase-treated type 1 GD patients by echocardiography, Doppler, and tissue Doppler echocardiography. This was a case-controlled study with 18 matched healthy volunteers. The obtained data was correlated with the levels of NT-proBNP (brain natriuretic peptide). None of the GD patients had clinical signs of heart Disease. In 3 of the 15 patients, we observed echocardiographic signs of aortic and mitral valve calcification. The left ventricular systolic function was within normal limits. Compared to the control group, there was no statistically significant difference observed in the most sensitive indices of left ventricular diastolic function, parameter Em (P=.095), and E/Em ratio (P=.097), as demonstrated by tissue Doppler echocardiography. However, there was a positive correlation between the E/Em ratio and NT-proBNP plasma levels (P=.009). In conclusion, the prospective echocardiographic study of type 1 GD patients did not validate any left ventricular dysfunction. But, the E/Em ratio showed a strong statistical correlation with the most sensitive indicators of heart failure, NT-proBNP. Research on larger groups of patients and the usage of even more sensitive methods as strain-rate imaging will be necessary to confirm eventual myocardial involvement in GD patients
Eapen Mathew - One of the best experts on this subject based on the ideXlab platform.
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bmr medicine Gauchers Disease a rare case report
BMR Medicine, 2016Co-Authors: Eapen Mathew, Uday Shankar SAbstract:Gaucher’s Disease is the most common group of lysosomal storage disorders caused by defective activity of an enzyme β- glucosidase leading to accumulation of glucocerebroside in cells of macrophage lineage. Accumulation of glucosylceramide in tissues leads to multisystem organ involvement. The "Gaucher cells" can be found in the spleen, liver, bone, and central nervous system in affected individuals, causing hepatosplenomegaly, anemia, thrombocytopenia and skeletal Diseases. Serum β glucosidase levels <15% of mean normal activity confirms the diagnosis, enzyme replacement being the only definitive treatment. We hereby report a case of Gauchers Disease type 1 or non-neuropathic form presented with pulmonary involvement. Despite its rarity in incidence, we presented this case to emphasize the importance of clinical examination and bone marrow finding in the diagnosis of G.D. Early diagnosis is important, because the Disease is rare and diagnosis may be delayed.
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case study Gauchers Disease a rare case report
2015Co-Authors: N C Shivaprakash, Eapen Mathew, B G NagaraAbstract:Gaucher’s Disease is the most common group of lysosomal storage disorders caused by defective activity of an enzyme βglucosidase leading to accumulation of glucocerebroside in cells of macrophage lineage. Accumulation of glucosylceramide in tissues leads to multisystem organ involvement. The "Gaucher cells" can be found in the spleen, liver, bone, and central nervous system in affected individuals, causing hepatosplenomegaly, anemia, thrombocytopenia and skeletal Diseases. Serum β glucosidase levels <15% of mean normal activity confirms the diagnosis, enzyme replacement being the only definitive treatment. We hereby report a case of Gauchers Disease type 1 or non-neuropathic form presented with pulmonary involvement. Despite its rarity in incidence, we presented this case to emphasize the importance of clinical examination and bone marrow finding in the diagnosis of G.D. Early diagnosis is important, because the Disease is rare and diagnosis may be delayed.
Uday Shankar S - One of the best experts on this subject based on the ideXlab platform.
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bmr medicine Gauchers Disease a rare case report
BMR Medicine, 2016Co-Authors: Eapen Mathew, Uday Shankar SAbstract:Gaucher’s Disease is the most common group of lysosomal storage disorders caused by defective activity of an enzyme β- glucosidase leading to accumulation of glucocerebroside in cells of macrophage lineage. Accumulation of glucosylceramide in tissues leads to multisystem organ involvement. The "Gaucher cells" can be found in the spleen, liver, bone, and central nervous system in affected individuals, causing hepatosplenomegaly, anemia, thrombocytopenia and skeletal Diseases. Serum β glucosidase levels <15% of mean normal activity confirms the diagnosis, enzyme replacement being the only definitive treatment. We hereby report a case of Gauchers Disease type 1 or non-neuropathic form presented with pulmonary involvement. Despite its rarity in incidence, we presented this case to emphasize the importance of clinical examination and bone marrow finding in the diagnosis of G.D. Early diagnosis is important, because the Disease is rare and diagnosis may be delayed.
B G Nagara - One of the best experts on this subject based on the ideXlab platform.
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case study Gauchers Disease a rare case report
2015Co-Authors: N C Shivaprakash, Eapen Mathew, B G NagaraAbstract:Gaucher’s Disease is the most common group of lysosomal storage disorders caused by defective activity of an enzyme βglucosidase leading to accumulation of glucocerebroside in cells of macrophage lineage. Accumulation of glucosylceramide in tissues leads to multisystem organ involvement. The "Gaucher cells" can be found in the spleen, liver, bone, and central nervous system in affected individuals, causing hepatosplenomegaly, anemia, thrombocytopenia and skeletal Diseases. Serum β glucosidase levels <15% of mean normal activity confirms the diagnosis, enzyme replacement being the only definitive treatment. We hereby report a case of Gauchers Disease type 1 or non-neuropathic form presented with pulmonary involvement. Despite its rarity in incidence, we presented this case to emphasize the importance of clinical examination and bone marrow finding in the diagnosis of G.D. Early diagnosis is important, because the Disease is rare and diagnosis may be delayed.