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Estudo das propriedades bioquímicas da enzima alfa-glicosidase ácida de pacientes com doença de Pompe em diferentes amostras biológicas : comparação com a enzima de indivíduos normaisMezzalira, Jamila January 2014 (has links)
A Doença de Pompe (DP), também conhecida como Doença de Armazenamento do Gligogênio Tipo II, é uma doença lisossômica de depósito (DLD) causada pela deficiência da enzima α-glicosidase ácida (GAA). A GAA catalisa a clivagem das ligações glicosídicas α-1,4 e α-1,6 da molécula de glicogênio, e sua deficiência gera um acúmulo intralisossomal de glicogênio em vários tecidos. Esse acúmulo é expressivo no tecido muscular, e com isso surge o aparecimento dos sintomas clínicos. Clinicamente, a DP manifesta-se através de um amplo espectro de fenótipos, que apresentam em comum a ocorrência de fraqueza muscular progressiva. O diagnóstico da DP é realizado através da medida da atividade enzimática em células sanguíneas e tecidos ou por análise da mutação gênica. O padrão-ouro para o diagnóstico é a medida da atividade da GAA em amostras de fibroblastos e, embora este tipo de diagnóstico seja definitivo, amostras de sangue também estão sendo utilizadas. A medida da atividade das enzimas lisossomais vem sendo atualmente realizada em amostras de sangue impregnado em papel filtro (SPF) como método de triagem neonatal e de populações de alto risco. Sabendo que a Terapia de Reposição Enzimática para DP já está disponível e melhora a sobrevida dos pacientes, o diagnóstico precoce da DP é essencial, deve ser adequado e de fácil acesso para que o tratamento inicie cedo e seja mais eficaz. Assim, este estudo teve como objetivo avaliar algumas caracteristicas bioquímicas e cinéticas da enzima em amostras de leucócitos totais e SPF de modo a observar as diferenças quanto ao seu comportamento, entre controles e pacientes, estabelecer valores de coeficientes de variação para as técnicas e observar o efeito do uso de diferentes concentrações do inibidor acarbose. A GAA de pacientes com DP mostrou um comportamento diferente do observado em controles saudáveis em termos dos parâmetros analisados (Km, Vmax, estabilidade térmica), em leucócitos totais. Em SPF, GAA mostrou um comportamento diferente do observado nos controles saudáveis em termos de pH ótimo e estabilidade térmica; e valores de Km e Vmax para GAA foram estabelecidos somente em SPF de controles saudáveis. O uso do inibidor acarbose é essencial para a análise enzimática da GAA em amostras de leucócitos totais e SPF por inibir seletivamente a isoenzima MGA e assim, garantir o diagnóstico da DP. Os valores de coeficiente de variação estabelecidos para as técnicas em leucócitos e SPF estão dentro do aceitável e expressam boa precisão e reprodutibilidade das técnicas. A técnica fluorimétrica usando o substrato 4- Metilumbeliferil-α-D-glicopiranosideo em leucócitos é confiável podendo ser utilizada para diagnóstico definitivo e padrão da Doença de Pompe; em SPF, pode contribuir para melhorar a triagem desta doença, diferenciando pacientes com PD de indivíduos normais, tornando o processo de investigação e diagnóstico mais preciso e confiável. / Pompe disease (PD), also known as Glycogen Storage Disease Type II, is a lysosomal storage disorder (LSD) caused by the deficiency of the acid α-glucosidase enzyme (GAA). GAA catalyzes the cleavage of the glycosidic bonds α-1,4 and α-1,6 of the glycogen molecule, and when GAA activity is deficient glycogen accumulates intralysosomally in several tissues. This accumulation is significant in muscle tissue, which leads to the onset of clinical symptoms. Clinically, PD is manifested through a wide escpectro phenotypes, which have in common the occurrence of progressive muscle weakness. PD is diagnosed by measuring GAA activity in blood cells and tissues or by gene mutation analysis. The gold standard for diagnosis is the measurement of GAA activity in fibroblast samples and although this diagnosis is definitive, blood samples are also being used. Lysosomal enzyme activity currently has been measured in dried blood spot (DBS) samples as a method for screening newborns and high-risk populations. Since enzyme replacement therapy is already available for PD and improves patient life expectancy, the early PD diagnosis is crucial and must be appropriate and easily available so that the treatment can be initiated early and be more effective. Thus, this study aimed to evaluate some biochemical and kinetic characteristics of the enzyme in samples of total leukocytes and DBS in order to observe the differences in behavior between controls and patients, establish values of coefficients of variation for the techniques and observe the effect of using different concentrations of inhibitor acarbose. GAA from PD patients showed a different behavior from that observed in healthy controls in terms of the analyzed parameters (Km, Vmax, thermal stability) in total leukocytes. In DBS, GAA showed a different behavior from that observed in healthy controls in terms of optimum pH and thermal stability. Km and Vmax values for GAA were established only in DBS healthy controls. The use of acarbose inhibitor is essential for enzymatic analysis of GAA in total leukocyte and DBS samples for selectively inhibiting MGA isozyme and thereby secures the diagnosis of PD. The coefficients of variation values established for techniques in leukocytes and DBS are acceptable, precise, and can be appropriately reproduced. The fluorimetric technique using 4- methylumbelliferyl-α-D-glucopyranoside substrate in leukocytes is reliable and can be used for definitive diagnosis of Pompe disease; in DBS, can help to improve the screening of this disease, differentiating PD patients from normal individuals, making the research process and more accurate and reliable diagnosis.
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Induction of autoantibodies to cathepsin L as a step towards an anti-cancer vaccine.Motsamai, Karabo. January 2005 (has links)
Cancer is a disease that is caused by mutations in somatic cells. Metastasis is the
major cause of death from cancer and often complicates treatment. Malignant
tumours secrete degradative enzymes such as cathepsin L which degrade the
extracellular matrix to facilitate tumour invasion and metastasis.
The immune system does not normally recognize and eradicate tumours because they
arise from self tissues to which the immune system is tolerant. Self antigens are
poorly immunogenic because they lack T cell help. In this study, a foreign glucosidase
was conjugated to self rabbit cathepsin L using glutaraldehyde to
specifically provide T helper cell epitopes. The conjugate was used to immunise two
male rabbits. A second pair of rabbits (male and female), was primed with sheep
cathepsin L (to induce T helper cell activation) and received rabbit cathepsin L
boosters. A third pair of rabbits which served as a control was immunised with sheep
cathepsin L. The two pairs of test rabbits made high avidity antibodies against rabbit
cathepsin L, showing a similar response to control rabbits when antibodies were
tested in an ELISA. Western blot analysis showed that these anti-cathepsin L
autoantibodies were specific for rabbit cathepsin L.
Rabbits which were immunised with the conjugate were · inoculated with sheep
cathepsin L nine weeks after the final inoculation with the conjugate. Analysis of
antibodies in an ELISA showed that antibody responses against rabbit cathepsin L
were augmented in a manner that is characteristic of memory responses. Low titre
antibodies against sheep cathepsin L were also produced. / Thesis (M.Sc.)-University of KwaZulu-Natal, Pietermaritzburg, 2005.
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The biosynthetic control of [alpha]-glucosidase and isomaltase in genetically defined strains of Saccharomyces cerevisiaeGorman, John William, January 1963 (has links)
Thesis (Ph. D.)--University of Wisconsin--Madison, 1963. / Vita. Includes: Comparison of the [alpha]-glucosides of Saccharomyces produced in response to five non-allelic maltose genes / Harlyn O. Halvorson, Sara Winderman and John Gorman. Reprinted from Biochimica et biophysica acta, vol. 67 (1963), p. 42-53 -- Relationship between protein and nucleic acid synthesis in Pseudomonas azotogensis grown in hexetidine / John Gorman and Harlyn Halvorson. Reprinted from Archives of biochemistry and biophysics, vol. 84, no. 2 (Oct. 1959), p. 462-470 -- The abnormal pattern of protein synthesis in Pseudomonas azotogensis in the presence of hexetidine / H.O. Halvorson and John Gorman. Reprinted from Experimental cell research, vol. 17, p. 522-524. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.
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The evaluation of β-glucosidase activity produced by wine-isolated yeastsPotgieter, Nydia, 1977- 03 1900 (has links)
Thesis (MSc)--University of Stellenbosch, 2004. / ENGLISH ABSTRACT: ~-Glucosidases constitute a major group of biologically important enzymes that
catalyze the hydrolysis of glycosidic linkages in ~-glucosides, as well as in glycosides
that contain only carbohydrate residues, e.g. cellobiose. These enzymes occur in all
living kingdoms and perform a variety of functions in organisms ranging from bacteria
to highly evolved mammals. Three different types of ~-glucosidases are found in
humans, each with its own function: glucocerebrosidase (a deficiency causes
Gaucher disease), lactase-phlorizin hydrolase (a deficiency results in lactose
intolerance) and cytosolic ~-glucosidase (responsible for the hydrolysis of ~-
glucosides ingested with foods of plant and animal origin). In plants, the functions of
~-glucosidases include pigment metabolism, biomass conversion and cyanogenesis,
a function it shares with insect ~-glucosidases. Microbial ~-glucosidases, as part of
the cellulase enzyme system that is responsible for the hydrolysis of cellobiose and
short-chain oligosaccharides into glucose, playa role in the conversion of cellulosic
biomass to liquid fuel. These microbial ~-glucosidases also playa very important role
in the enhancement of fruit and wine aromas through the liberation of monoterpenols.
Monoterpenols play an invaluable role in the flavor and aroma of grapes and
wine, and are present as free, volatile and odorous molecules, as well as flavorless,
non-volatile glycosidic complexes. These complexes most often occur as 6-0-~-Dxvlopyranosyl-
B-Dcqlucopyranosides, 6-0-~-D-glucopyranosyl-~-D-glucopyranosides,
6-0-a-L-arabinofuranosyl-~-D-glucopyranosides, 6-0-a-L-rhamnopyranosyl-~-Dglucopyranosides,
or 6-0-~-D-apiofuranosyl-~-D-glucopyranosides of mainly linalool,
geraniol, nerol, a-terpineol and hotrienol. Two mechanisms exist for the release of
monoterpenes from glycosidically bound non-volatile precursors: acid hydrolysis and
enzymatic hydrolysis. As high temperature acid hydrolysis causes a rearrangement
of the monoterpene aglycones, the focus has shifted towards the more efficient
enzymatic hydrolysis that does not result in modifications of the intrinsic aromatic
character of the wine.
The endogenous ~-glucosidases of grapes (Vitis vinifera), as well as of the wine
yeast Saccharomyces cerevisiae, exhibit very low activity towards the glycoside
precursors, and thus the focus has increasingly fallen on the addition of exogenous
~-glucosidases to enhance wine flavor. Fungal, bacterial and some yeast ~-
glucosidases have been indicated as effective aroma liberators, but these enzymes
are not always suitable for use under the harsh conditions that prevail during
winemaking (i.e. low pH, low temperatures, and high ethanol and glucose
concentrations). The limited enzyme activities of the abovementioned
microorganisms have resulted in a search among non-Saccharomyces yeasts for ~-
glucosidases that can withstand these conditions.
The ~-glucosidase activities of 20 wine-associated non-Saccharomyces yeasts
were quantified, characterized and assessed to determine the efficiency with which
they could liberate monoterpenols from their terpenyl-glycosides. The Debaryomyces pseudopolymorphus l3-glucosidase from intracellular crude cell extracts exhibited the
most suitable combination of properties in terms of functionality at wine pH,
resistance to wine-associated inhibitory compounds (glucose, ethanol and sulfur
dioxide), high substrate affinity and large aglycone-substrate recognition. This yeast
strain was also used, in conjunction with S. cerevisiae VIN13, for the small-scale
fermentation of Chardonnay juice. The results indicated that the l3-glucosidase of D.
pseudopolymorphus had definite potential as a wine aroma-enhancing enzyme, as
the concentrations of free terpenols (nerol, geraniol and citronellol) were significantly
increased during fermentation.
Future experimental work would include an in-depth study of the kinetic
characteristics of the l3-glucosidases (both cytosolic and cell-associated) exhibiting
the highest terpenol-liberating activity under winemaking conditions. The next step
would then be the cloning and expression of the most efficient l3-glucosidase gene in
a commercial wine yeast. Such a recombinant wine yeast would release grapederived
aroma compounds from their non-volatile precursors during single culture
fermentations, thereby increasing the sensorial quality of wine. / AFRIKAANSE OPSOMMING: I3-Glukosidases vorm deel van 'n groot groep biologies belangrike ensieme wat die
hidrolise van glikosidiese bindings binne l3-glukosiede,sowel as binne glikosiede wat
slegs uit koolhidraatresidue bestaan, soos bv. sellobiose, kataliseer. Hierdie ensieme
kom in alle koningkryke van lewende organismes voor en verrig 'n wye
verskeidenheid funksies binne organismes wat wissel van bakterieë tot hoogs
ontwikkelde soogdiere. Drie verskillende tipes l3-glukosidases,elk met sy eie funksie,
kom in mense voor: glukoserebrosidase ('n gebrek hieraan lei tot Gaucher-siekte),
laktaseflorizinhidrolase ('n gebrek hieraan gee aanleiding tot laktose-intoleransie) en
sitosol l3-glukosidase (verantwoordelik vir die hidrolise van l3-glukosiede wat saam
met voedsel van plant en dier oorsprong ingeneem word). Die funksies van 13-
glukosidase binne plante sluit in pigmentmetabolisme, biomassa-omsetting en
sianogenese, wat ook 'n funksie van insek l3-glukosidases is. Mikrobiese 13-
glukosidases, as deel van die sellulase-ensiemsisteem wat verantwoordelik vir die
hidrolise van sellobiose en kortketting-oligosakkariede na glukose is, speel 'n rol in
die omsetting van sellulosebiomassa na brandstof. Hierdie mikrobiese 13-
glukosidases speelook 'n baie belangrike rol in die verbetering van vrugte- en
wynaroma deur die vrystelling van monoterpenole.
Monoterpenole speel 'n belangrike rol in die geur en aroma van druiwe en wyn,
en kom voor as vry, vlugtige en aromatiese molekules, asook geurlose, nie-vlugtige
glikosidies-gebonde komplekse. Hierdie komplekse is meestal in die vorm van 6-0-
I3-D-xilopiranosiel-I3-D-glukopiranosiede, 6-0-a-L-arabinofuranosiel-I3-D-glukopiranosiede,
6-0-I3-D-glukopiranosiel-I3-D-glukopiranosiede, 6-0-a-L-ramnopiranosiel-
I3-D-glukopiranosiede,of 6-0-I3-D-apiofuranosiel-I3-D-glukopiranosiedevan
hoofsaaklik linalool, geraniol, nerol, a-terpineol en hotrienol. Monoterpenole kan op
een van twee maniere van hul suikermolekules vrygestel word: suurhidrolise of
ensimatiese hidrolise. Die hoë temperature waarby suurhidrolise plaasvind
veroorsaak 'n herrangskikking van die monoterpeen aglikone, en die fokus het
gevolglik verskuif na die meer effektiewe ensimatiese hidrolise wat nie verandering
van die intrinsieke aromatiese karakter van die wyn tot gevolg het nie.
Die endogene l3-glukosidases van druiwe (Vitis vinifera) en die wyngis
Saccharomyces cere visiae , toon baie lae aktiwiteit ten opsigte van die aromatiese
voorlopers, en dus word daar toenemend op die toevoeging van eksogene 13-
glukosidases tot die wyn gefokus om meer geur vry te stel. Daar is bevind dat 13-
glukosidases van fungiese, bakteriële en gis oorsprong effektiewe aromavrystelIers
is, maar hierdie ensieme is nie altyd gepas vir gebruik in wyn nie, aangesien dit 'n
omgewing is met 'n lae pH, lae temperatuur, en hoë etanol- en glukosekonsentrasies.
Die beperkte ensiemaktiwitiet van bogenoemde mikroorganismes het gelei tot 'n
soeke onder nie-Saccharomyces giste na l3-glukosidases wat in die wynomgewing
kan funksioneer. Die ~-glukosidase-aktiwiteit van twintig wyn geassosieerde nie-Saccharomyces
giste is gekwantifiseer en gekarakteriseer om te bepaal tot watter mate dit
monoterpenole van hul terpeniel glikosiede kan vrystel. Die intrasellulêre ~-
glukosidase teenwoordig in the selekstrak van Debaryomyces pseudopolymorphus,
het die belowendste resultate getoon ten opsigte van funksionaliteit by wyn se pH,
weerstand teen wyn geassosieerde inhibeerders (glukose, etanol en
swaweidioksied), hoë substraataffiniteit en breë aglikoon-substraat herkenning.
Hierdie gisras is ook in kombinasie met S. cerevisiae VIN13 gebruik vir die
kleinskaalse fermentasie van Chardonnay sap. Die resultate het getoon dat die ~-
glukosidase van D. pseudopolymorphus wel potensiaal het om wynaroma te
verhoog, aangesien die konsentrasie van ongebonde terpenole (nerol, geraniol en
citronellol) aansienlik tydens fermentasie toegeneem het.
Toekomstige eksperimentele werk sluit in, onder meer, In in-diepte studie van die
kinetiese eienskappe van die ~-glukosidases (beide sitesolies en sel-geassosieerd)
wat die meeste terpenole onder wynrnaakkondisies vry stel, asook die klonering en
uitdrukking van die enkele ~-glukosidasegeen met die hoogste aktiwiteit, in In
kommersiële wyngis. Só In rekombinante wyngis sal die vrystelling van druifgebaseerde
aromakomponente van hul nie-vlugtige, geurlose voorlopers tydens
enkel-kultuur fermentasies teweeg bring.
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Estudo das propriedades bioquímicas da enzima alfa-glicosidase ácida de pacientes com doença de Pompe em diferentes amostras biológicas : comparação com a enzima de indivíduos normaisMezzalira, Jamila January 2014 (has links)
A Doença de Pompe (DP), também conhecida como Doença de Armazenamento do Gligogênio Tipo II, é uma doença lisossômica de depósito (DLD) causada pela deficiência da enzima α-glicosidase ácida (GAA). A GAA catalisa a clivagem das ligações glicosídicas α-1,4 e α-1,6 da molécula de glicogênio, e sua deficiência gera um acúmulo intralisossomal de glicogênio em vários tecidos. Esse acúmulo é expressivo no tecido muscular, e com isso surge o aparecimento dos sintomas clínicos. Clinicamente, a DP manifesta-se através de um amplo espectro de fenótipos, que apresentam em comum a ocorrência de fraqueza muscular progressiva. O diagnóstico da DP é realizado através da medida da atividade enzimática em células sanguíneas e tecidos ou por análise da mutação gênica. O padrão-ouro para o diagnóstico é a medida da atividade da GAA em amostras de fibroblastos e, embora este tipo de diagnóstico seja definitivo, amostras de sangue também estão sendo utilizadas. A medida da atividade das enzimas lisossomais vem sendo atualmente realizada em amostras de sangue impregnado em papel filtro (SPF) como método de triagem neonatal e de populações de alto risco. Sabendo que a Terapia de Reposição Enzimática para DP já está disponível e melhora a sobrevida dos pacientes, o diagnóstico precoce da DP é essencial, deve ser adequado e de fácil acesso para que o tratamento inicie cedo e seja mais eficaz. Assim, este estudo teve como objetivo avaliar algumas caracteristicas bioquímicas e cinéticas da enzima em amostras de leucócitos totais e SPF de modo a observar as diferenças quanto ao seu comportamento, entre controles e pacientes, estabelecer valores de coeficientes de variação para as técnicas e observar o efeito do uso de diferentes concentrações do inibidor acarbose. A GAA de pacientes com DP mostrou um comportamento diferente do observado em controles saudáveis em termos dos parâmetros analisados (Km, Vmax, estabilidade térmica), em leucócitos totais. Em SPF, GAA mostrou um comportamento diferente do observado nos controles saudáveis em termos de pH ótimo e estabilidade térmica; e valores de Km e Vmax para GAA foram estabelecidos somente em SPF de controles saudáveis. O uso do inibidor acarbose é essencial para a análise enzimática da GAA em amostras de leucócitos totais e SPF por inibir seletivamente a isoenzima MGA e assim, garantir o diagnóstico da DP. Os valores de coeficiente de variação estabelecidos para as técnicas em leucócitos e SPF estão dentro do aceitável e expressam boa precisão e reprodutibilidade das técnicas. A técnica fluorimétrica usando o substrato 4- Metilumbeliferil-α-D-glicopiranosideo em leucócitos é confiável podendo ser utilizada para diagnóstico definitivo e padrão da Doença de Pompe; em SPF, pode contribuir para melhorar a triagem desta doença, diferenciando pacientes com PD de indivíduos normais, tornando o processo de investigação e diagnóstico mais preciso e confiável. / Pompe disease (PD), also known as Glycogen Storage Disease Type II, is a lysosomal storage disorder (LSD) caused by the deficiency of the acid α-glucosidase enzyme (GAA). GAA catalyzes the cleavage of the glycosidic bonds α-1,4 and α-1,6 of the glycogen molecule, and when GAA activity is deficient glycogen accumulates intralysosomally in several tissues. This accumulation is significant in muscle tissue, which leads to the onset of clinical symptoms. Clinically, PD is manifested through a wide escpectro phenotypes, which have in common the occurrence of progressive muscle weakness. PD is diagnosed by measuring GAA activity in blood cells and tissues or by gene mutation analysis. The gold standard for diagnosis is the measurement of GAA activity in fibroblast samples and although this diagnosis is definitive, blood samples are also being used. Lysosomal enzyme activity currently has been measured in dried blood spot (DBS) samples as a method for screening newborns and high-risk populations. Since enzyme replacement therapy is already available for PD and improves patient life expectancy, the early PD diagnosis is crucial and must be appropriate and easily available so that the treatment can be initiated early and be more effective. Thus, this study aimed to evaluate some biochemical and kinetic characteristics of the enzyme in samples of total leukocytes and DBS in order to observe the differences in behavior between controls and patients, establish values of coefficients of variation for the techniques and observe the effect of using different concentrations of inhibitor acarbose. GAA from PD patients showed a different behavior from that observed in healthy controls in terms of the analyzed parameters (Km, Vmax, thermal stability) in total leukocytes. In DBS, GAA showed a different behavior from that observed in healthy controls in terms of optimum pH and thermal stability. Km and Vmax values for GAA were established only in DBS healthy controls. The use of acarbose inhibitor is essential for enzymatic analysis of GAA in total leukocyte and DBS samples for selectively inhibiting MGA isozyme and thereby secures the diagnosis of PD. The coefficients of variation values established for techniques in leukocytes and DBS are acceptable, precise, and can be appropriately reproduced. The fluorimetric technique using 4- methylumbelliferyl-α-D-glucopyranoside substrate in leukocytes is reliable and can be used for definitive diagnosis of Pompe disease; in DBS, can help to improve the screening of this disease, differentiating PD patients from normal individuals, making the research process and more accurate and reliable diagnosis.
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Estudo das propriedades bioquímicas da enzima alfa-glicosidase ácida de pacientes com doença de Pompe em diferentes amostras biológicas : comparação com a enzima de indivíduos normaisMezzalira, Jamila January 2014 (has links)
A Doença de Pompe (DP), também conhecida como Doença de Armazenamento do Gligogênio Tipo II, é uma doença lisossômica de depósito (DLD) causada pela deficiência da enzima α-glicosidase ácida (GAA). A GAA catalisa a clivagem das ligações glicosídicas α-1,4 e α-1,6 da molécula de glicogênio, e sua deficiência gera um acúmulo intralisossomal de glicogênio em vários tecidos. Esse acúmulo é expressivo no tecido muscular, e com isso surge o aparecimento dos sintomas clínicos. Clinicamente, a DP manifesta-se através de um amplo espectro de fenótipos, que apresentam em comum a ocorrência de fraqueza muscular progressiva. O diagnóstico da DP é realizado através da medida da atividade enzimática em células sanguíneas e tecidos ou por análise da mutação gênica. O padrão-ouro para o diagnóstico é a medida da atividade da GAA em amostras de fibroblastos e, embora este tipo de diagnóstico seja definitivo, amostras de sangue também estão sendo utilizadas. A medida da atividade das enzimas lisossomais vem sendo atualmente realizada em amostras de sangue impregnado em papel filtro (SPF) como método de triagem neonatal e de populações de alto risco. Sabendo que a Terapia de Reposição Enzimática para DP já está disponível e melhora a sobrevida dos pacientes, o diagnóstico precoce da DP é essencial, deve ser adequado e de fácil acesso para que o tratamento inicie cedo e seja mais eficaz. Assim, este estudo teve como objetivo avaliar algumas caracteristicas bioquímicas e cinéticas da enzima em amostras de leucócitos totais e SPF de modo a observar as diferenças quanto ao seu comportamento, entre controles e pacientes, estabelecer valores de coeficientes de variação para as técnicas e observar o efeito do uso de diferentes concentrações do inibidor acarbose. A GAA de pacientes com DP mostrou um comportamento diferente do observado em controles saudáveis em termos dos parâmetros analisados (Km, Vmax, estabilidade térmica), em leucócitos totais. Em SPF, GAA mostrou um comportamento diferente do observado nos controles saudáveis em termos de pH ótimo e estabilidade térmica; e valores de Km e Vmax para GAA foram estabelecidos somente em SPF de controles saudáveis. O uso do inibidor acarbose é essencial para a análise enzimática da GAA em amostras de leucócitos totais e SPF por inibir seletivamente a isoenzima MGA e assim, garantir o diagnóstico da DP. Os valores de coeficiente de variação estabelecidos para as técnicas em leucócitos e SPF estão dentro do aceitável e expressam boa precisão e reprodutibilidade das técnicas. A técnica fluorimétrica usando o substrato 4- Metilumbeliferil-α-D-glicopiranosideo em leucócitos é confiável podendo ser utilizada para diagnóstico definitivo e padrão da Doença de Pompe; em SPF, pode contribuir para melhorar a triagem desta doença, diferenciando pacientes com PD de indivíduos normais, tornando o processo de investigação e diagnóstico mais preciso e confiável. / Pompe disease (PD), also known as Glycogen Storage Disease Type II, is a lysosomal storage disorder (LSD) caused by the deficiency of the acid α-glucosidase enzyme (GAA). GAA catalyzes the cleavage of the glycosidic bonds α-1,4 and α-1,6 of the glycogen molecule, and when GAA activity is deficient glycogen accumulates intralysosomally in several tissues. This accumulation is significant in muscle tissue, which leads to the onset of clinical symptoms. Clinically, PD is manifested through a wide escpectro phenotypes, which have in common the occurrence of progressive muscle weakness. PD is diagnosed by measuring GAA activity in blood cells and tissues or by gene mutation analysis. The gold standard for diagnosis is the measurement of GAA activity in fibroblast samples and although this diagnosis is definitive, blood samples are also being used. Lysosomal enzyme activity currently has been measured in dried blood spot (DBS) samples as a method for screening newborns and high-risk populations. Since enzyme replacement therapy is already available for PD and improves patient life expectancy, the early PD diagnosis is crucial and must be appropriate and easily available so that the treatment can be initiated early and be more effective. Thus, this study aimed to evaluate some biochemical and kinetic characteristics of the enzyme in samples of total leukocytes and DBS in order to observe the differences in behavior between controls and patients, establish values of coefficients of variation for the techniques and observe the effect of using different concentrations of inhibitor acarbose. GAA from PD patients showed a different behavior from that observed in healthy controls in terms of the analyzed parameters (Km, Vmax, thermal stability) in total leukocytes. In DBS, GAA showed a different behavior from that observed in healthy controls in terms of optimum pH and thermal stability. Km and Vmax values for GAA were established only in DBS healthy controls. The use of acarbose inhibitor is essential for enzymatic analysis of GAA in total leukocyte and DBS samples for selectively inhibiting MGA isozyme and thereby secures the diagnosis of PD. The coefficients of variation values established for techniques in leukocytes and DBS are acceptable, precise, and can be appropriately reproduced. The fluorimetric technique using 4- methylumbelliferyl-α-D-glucopyranoside substrate in leukocytes is reliable and can be used for definitive diagnosis of Pompe disease; in DBS, can help to improve the screening of this disease, differentiating PD patients from normal individuals, making the research process and more accurate and reliable diagnosis.
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Molecular Modeling: Elucidation of Structure/Function Relationships of Proteins and DNA at the Atomic ResolutionRuscio, Jory Zmuda 08 May 2007 (has links)
While experiments provide valuable information about biological molecules, current technology cannot yet monitor atomic fluctuations at relevant time scales. Theoretical computational simulations are able to model the appropriate interactions at atomic resolution. Computational techniques have become widely used for identifying interactions in biological systems. Such methods have proven quite accurate in their ability to reproduce experimental data and also in screening and predicting pertinent activities. Molecular modeling employs theoretical and computational techniques to elucidate biologically relevant information from macromolecular structures. Three biological systems, the nucleosome core particle, myoglobin and glycosyl hydrolase family 1 beta-glucosidases will be examined with molecular modeling methods. Results of our analyses provide information about DNA flexibility and packaging, internal migration of ligands in a small protein, and substrate specificity of an enzyme system. / Ph. D.
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Síntese e avaliação biológica de glycoclusters com potencial aplicação para o tratamento de diabetes e doença de Chagas / Synthesis and biological evaluation of glycoclusters with potential applications in diabetes and Chagas diseaseFiguerêdo, Andreza da Silva 02 April 2018 (has links)
As funções abrangentes e complexas de carboidratos nos sistemas biológicos oferecem muitas possibilidades para explorar essas estruturas no desenvolvimento de estratégias terapêuticas. Como consequência de sua estrutura e função, algumas glicosidases e glicosiltransferases envolvidas em patologias significativas como diabetes e doença de Chagas são alvos interessantes na busca por inibidores enzimáticos baseados em carboidratos. Assim, o conhecimento de características estruturais e mecanismo de ação de ?- e ?-glucosidases e trans-sialidases e as sólidas evidências de que glicosidases são sensíveis a efeito de multivalência sustentaram o planejamento de glycoclusters potencialmente inibidores dessas enzimas contendo três, quatro ou seis unidades correlatas aos seus substratos naturais (glicose e galactose/ácido siálico). Adicionalmente foram propostos glycoclusters baseados na estrutura do glicoaminoácido ?-GalNAc-O-Thr. Empregando reações de cicloadição CuAAC, treze dos dezesseis glycoclusters tetra- e hexavalentes inicialmente propostos foram obtidos em bons rendimentos, sendo 8 derivados de glicose, 4 de galactose e o derivado tetravalente de ácido siálico 7. Dez desses compostos são inéditos na literatura. Com a finalidade de gerar diversidade estrutural o derivado trivalente 29 foi preparado e permitiu o acoplamento de um imino-açúcar D-gluco ou L-gulo DNJ a estruturas trivalentes de glicose. A mesma estratégia levou à obtenção do glycocluster 85, contendo unidades de galactose e um resíduo de ácido siálico. Ensaios de cinética em ?-glucosidases de arroz e Saccharomyces cerevisiae e ?-glucosidase de amêndoas com os glycoclusters derivados de glicose 1-4, 9-12 e 63-66 mostraram atividade interessante apenas para os compostos mistos, evidenciando a importância do imino-açúcar para a interação com essas enzimas. No que diz respeito aos derivados multivalentes de galactose e/ou ácido siálico 5-7, 13, 14 e 85, inibição expressiva da enzima TcTS foi alcançada pelo derivado 7 (IC50 450 ?M). Ensaios de atividade tripanocida, bloqueio de invasão e citotoxicidade sobre fibroblastos de mamífero não-infectados cultivados in vitro evidenciaram atividade de todos esses compostos contra a forma amastigota e bloqueio de invasão celular por T. cruzi (diminuindo a infecção em concentrações de até 25?M). / The comprehensive and elaborate functions of carbohydrates in biological systems offer countless possibilities to apply these structures in the development of therapeutic strategies. Because of their structure and function, some glycosidases and glycosyltransferases involved in pathological conditions such as diabetes and Chagas disease are interesting targets in the search for carbohydrate-based enzyme inhibitors. Thus, the knowledge of structural features and the mechanism of action of ?- and ?-glucosidases and trans-sialidases, combined with strong evidences that glycosidases are sensitive to multivalent effect, have supported the design of substrate-based glycoclusters with potential glycosidases inhibitory properties. In addition, glycoclusters based on the structure of the ?-GalNAc-O-Thr glycoamino acid have been planned. Using CuAAC cycloaddition reactions, thirteen of the sixteen tetra- and hexavalent glycoclusters were obtained in good yields, being 8 glucose derivatives, 4 galactose, and the tetravalent sialic acid derivative 7. Ten of these compounds are unpublished. In order to generate structural diversity, the trivalent block 29 was prepared and allowed the coupling of trivalent glucose clusters to iminosugars D-gluco or L-gulo DNJ isomers. The same strategy afforded glycocluster 85, containing galactose units and a sialic acid residue. Kinetic assays in ? and ?-glucosidases with the glucose-derived glycoclusters 1-4, 9-12 and 63-66 showed interesting activity only for the heteroclusters compounds, evidencing the relevance of iminosugars for the interaction with these enzymes. Regarding galactose and/or sialic acid cluster 5-7, 13, 14 and 85, significant inhibition of the TcTS enzyme was achieved by derivative 7 (IC50 450 ?M). In vitro assays for trypanocidal activity and cytotoxicity showed good results for of all these compounds. They appear to block host cell invasion by T. cruzi (reducing infection index in concentrations up to 25?M).
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Études structurales et fonctionnelles d'alpha-glucosidases bactériennes / Functional and structural studies of bacterial alpha-glucosidasesDejob, Magali 15 July 2013 (has links)
Il est reconnu, depuis des années, que la flore intestinale par son équilibre complexe et dynamique joue un rôle essentiel dans la santé humaine. Une des stratégies les plus prometteuses pour la maintenir ou l’améliorer consiste à moduler le microbiome par l’utilisation de bactéries probiotiques ou de sucres prébiotiques. C’est dans ce contexte que s’inscrivent les études structurales et fonctionnelles d’α glucosidases bactériennes développées dans cette thèse. Ces enzymes hydrolysant les liaisons α-(1,4) glucosidiques sont classées, selon la base de données CAZy, dans les familles de glycoside hydrolases (GH) 4, 13, 31, 63, 97 et 122. Ces travaux de thèse, centrés sur trois α-glucosidases issues de Lactobacillus bulgaricus (11842aglu, GH31), Lactococcus lactis (1403aglu, GH13) et Shewanella sp. ANA-3 (SHWaglu, GH97), exposent la mise au point de leurs protocoles de surexpression et de purification. Ils présentent également des études bioinformatiques de 11842aglu et de 1403aglu, ainsi qu’une caractérisation enzymatique préliminaire de cette dernière. Une analyse structurale et fonctionnelle approfondie de SHWaglu a aussi été réalisée. La résolution, par cristallographie aux rayons X, des structures de SHWaglu seule, en complexe avec différents ligands et de mutants, a participé à enrichir les connaissances, jusqu’à présent peu étendues, sur les enzymes de la famille GH97. Ainsi, un motif structural conservé au sein de cette famille a notamment été mis en évidence. Par ailleurs, ces informations structurales combinées aux études enzymatiques ont permis de révéler des déterminants moléculaires de l’activité de cette α-glucosidase et, par conséquent, d’établir les relations structure-fonction-activité de cette enzyme. Ainsi, l’ensemble des données obtenues, couplé à des études d’ingénierie protéique, contribue à ouvrir de nouvelles perspectives industrielles, notamment en suggérant d’optimiser ou de conférer des activités enzymatiques modifiées dans certaines cibles de choix afin de leur faire synthétiser des sucres de type prébiotiques / It is now generally accepted that the gut flora with its complex and dynamic nature plays a vital role in human health. One of the most promising strategies for maintaining or improving health is to modulate the microbiome by the use of probiotics and prebiotics as food supplements. The structure/function/activity relationship studies of bacterial α-glucosidases described in this thesis have been performed within this context. These α-(1,4)-glucosidic bond hydrolyzing enzymes are classified, according to the CAZy database, into glycoside hydrolases families (GH) 4, 13, 31, 63, 97 and 122. This thesis work, has focused on three α-glucosidases from Lactobacillus bulgaricus (11842aglu, GH31), Lactococcus lactis (1403aglu, GH13) and Shewanella sp. ANA-3 (SHWaglu, GH97), and the development of their overexpression and purification protocols. It also presents a bioinformatics studies of 11842aglu and 1403aglu, as well as preliminary enzymatic characterization of the latter. As for SHWaglu, detailed structural and functional studies have been carried out. The crystal structures of SHWaglu in its native state, in complex with different ligands as well as site directed mutants have contributed to increase our knowledge on enzymes from the GH97 family which to date remains relatively limited. Notably, a conserved structural motif in this family has been identified. Overall, the structural- and enzymatic studies and analyses have revealed molecular-and structural determinants governing the activity and broad substrate specificity of this α-glucosidase which is adapted to cold temperatures. Apart from the insight gained from a fundamental research point of view, data described within this work, coupled with protein engineering studies may contribute to open up new industrial perspectives, in particular by suggesting optimized or altered enzyme activities in some attractive enzyme targets with the aim of synthesizing prebiotic compounds
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Expression of fungal b-glucosidases in Saccharomyces cerevisiae for enhanced growth on cellobioseNjokweni, Anathi Perseverence 12 1900 (has links)
Thesis (MSc (Microbiology))--Stellenbosch University, 2011. / ENGLISH ABSTRACT: Bio-fuels have been considered an ideal substitute for fossil fuels due to their availability and renewable nature. Bio-ethanol is currently of great market interest as an alternative fuel with the potential of supplementing petroleum as transportation fuel. Lignocellulosic biomass, a renewable energy source, can be "readily" converted to bio-ethanol. The main impediment in the conversion process is the recalcitrance of the main lignocellulosic components (cellulose, hemicelluloses and lignin) to enzymatic hydrolysis as well as the lack of available low-cost technology. Consolidated Bioprocessing (CBP) is a single process step which offers a cost-effective and economically feasible strategy for bio-ethanol production. The process requires micro-organisms that produce ethanol at high rates and titres.
Saccharomyces cerevisiae has potential as a CBP candidate due to its high ethanol yield, robustness in industrial processes, well-developed gene expression system and its safety status. Unfortunately S. cerevisiae does not degrade polysaccharides and therefore requires heterologous expression of cellulases. Genetic engineering of S. cerevisiae for cellulose hydrolysis serves as an important step in yeast strain development for CBP, and serves as a stepping stone for the commercialisation of lignocellulosic bio-ethanol. Although cellulose- utilising S. cerevisiae strains have been constructed, the cellobiose conversion is slow, hampering optimal ethanol production. β-glucosidases have been shown to be the major rate-limiting factors in cellulose saccharification as their activity determines the extent of cellulose hydrolysis, by removing excess cellobiose which causes feed-back inhibition on endoglucanase and cellobiohydrolase activities (Du Plessis et al. 2009;Lynd et al. 2002). Therefore, insufficient supply of β-glucosidase activity is detrimental to CBP and can be addressed by increasing the enzyme supply or using highly active β-glucosidases to enhance cellobiose hydrolysis.
In this study, several cellobiose fermenting S. cerevisiae strains were constructed. Extracellular fungal β-glucosidase-encoding genes were successfully expressed in S. cerevisiae under the transcriptional control of the ENO1 (enolase) promoter and terminator sequences. The recombinant enzymes produced were characterised based on pH and temperature optima as well as kinetic parameters. Bio-fuels have been considered an ideal substitute for fossil fuels due to their availability and renewable nature. Bio-ethanol is currently of great market interest as an alternative fuel with the potential of supplementing petroleum as transportation fuel. Lignocellulosic biomass, a renewable energy source, can be „readily‟ converted to bio-ethanol. The main impediment in the conversion process is the recalcitrance of the main lignocellulosic components (cellulose, hemicelluloses and lignin) to enzymatic hydrolysis as well as the lack of available low-cost technology. Consolidated Bioprocessing (CBP) is a single process step which offers a cost-effective and economically feasible strategy for bio-ethanol production. The process requires micro-organisms that produce ethanol at high rates and titres.
Saccharomyces cerevisiae has potential as a CBP candidate due to its high ethanol yield, robustness in industrial processes, well-developed gene expression system and its safety status. Unfortunately S. cerevisiae does not degrade polysaccharides and therefore requires heterologous expression of cellulases. Genetic engineering of S. cerevisiae for cellulose hydrolysis serves as an important step in yeast strain development for CBP, and serves as a stepping stone for the commercialisation of lignocellulosic bio-ethanol. Although cellulose- utilising S. cerevisiae strains have been constructed, the cellobiose conversion is slow, hampering optimal ethanol production. β-glucosidases have been shown to be the major rate-limiting factors in cellulose saccharification as their activity determines the extent of cellulose hydrolysis, by removing excess cellobiose which causes feed-back inhibition on endoglucanase and cellobiohydrolase activities (Du Plessis et al. 2009;Lynd et al. 2002). Therefore, insufficient supply of β-glucosidase activity is detrimental to CBP and can be addressed by increasing the enzyme supply or using highly active β-glucosidases to enhance cellobiose hydrolysis.
In this study, several cellobiose fermenting S. cerevisiae strains were constructed. Extracellular fungal β-glucosidase-encoding genes were successfully expressed in S. cerevisiae under the transcriptional control of the ENO1 (enolase) promoter and terminator sequences. The recombinant enzymes produced were characterised based on pH and temperature optima as well as kinetic parameters. / AFRIKAANSE OPSOMMING: Biobrandstof word beskou as die ideale plaasvervanger vir fossielbrandstof weens die beskikbaarheid en herwinbare aard daarvan. Bio-etanol wek tans groot mark-verwante belangstelling as alternatiewe brandstof weens die potensiaal om petroleum as vervoerbrandstof aan te vul. Lignosellulose biomassa, 'n hernubare energiebron, kan "maklik" tot bio-etanol omgeskakel word. Die groot struikelblok in die omskakelingsproses is die weerstandbiedendheid van die lignosellulose komponente (sellulose, hemisellulose en lignien) teen ensiematiese hidroliese asook die gebrek aan beskikbaarheid van lae koste tegnologie. Gekonsolideerde Bioprosessering (KBP) is 'n enkel stap proses wat 'n koste-effektiewe en ekonomiesvatbare strategie voorstel vir bio-etanolproduksie. Die proses benodig 'n mikroorganisme wat daartoe instaat is om etanol teen hoë vlakke en tempo te kan produseer.
Saccharomyces cerevisiae het potensiaal as 'n KBP kandidaat weens sy hoë vlakke van etanolproduksie, gehardheid in industriële prosesse, goed-ontwikkelde geenuitdrukking sisteme en veiligheidstatus. Ongelukkig kan S. cerevisiae nie polisakkariede afbreek nie en benodig derhalwe heteroloë uitdrukking van sellulases. Die genetiese manipulering van S. cerevisiae vir sellulose hidroliese dien as 'n belangrike stap in gisrasontwikkeling vir KBP en dien as 'n “stepping stone” vir die kommersialisasie van lignosellulose bio-etanol. Alhoewel sellulose-benuttende S. cerevisiae rasse reeds gekonstrueer is, is sellulose omskakeling stadig en belemmer dit optimale etanolproduksie. 'n Hoogs aktiewe glukosidase word derhalwe benodig om die hidroliese van sellobiose te versnel.
Die studie behels die konstruksie van verskeie sellobiose-fermenterende S. cerevisiae rasse. Ektrasellulêre, fungiese -glukosidase-koderende gene was suksesvol in S. cerevisiae uitgedruk onderhewig aan die transkripsionele beheer van die ENO1 (enulase) promoter en termineerder DNS-volgordes. Die geproduseerde, rekombinante ensieme is gekarakteriseer op grond van optimale pH en temperatuur, asook kinetiese eienskappe. Die intrasellulêre benutting van sellobiose is 'n ideale benadering tot sellobiose hidroliese siende dat dit die risiko van kontaminasie verminder wat veroorsaak word deur die glukose wat vrygestel word in die ekstrasellulêre omgewing. Tog beskik S. cerevisiae nie oor 'n vervoer meganisme om sellobiose in die sel in te bring nie. Derhalwe is die intrasellulêre Phanaerochaete chrysosporium -glukosidase-koderende geen suksesvol saam met die Kluyveromyces lactis laktose permease uitgedruk. Alle rekombinante rasse is vir groei op sellobiose geevalueer. Die mees belowendste esktrasellulêre -glukosidase-produserende S. cerevisiae Y294[Pccbgl1] ras toon 'n aktiwiteit van 3.85 nkat.g-1, 1.85 keer meer die aktiwiteit van die S. cerevisiae Y294[SFB] ras (2.07 nkat.g-1). S. cerevisiae Y294[Pccbgl1] het ook 'n maksimum groei tempo van 0.25 h-1 onder anearobiese kondisies in vergelyking met die 0.064 h-1 van S. cerevisiae Y294[iPcbglB+lac12] toon. Onder anaërobe kondisies het S. cerevisiae Y294[Pccbgl1] 7.95 g.l-1 sellobiose verbruik en 4.05 g. l-1 etanol geproduseer oor 'n tydperk van 116 uur, terwyl S. cerevisiae Y294[iPcbglB+lac12] 0.41 g.l-1 sellobiose verbruik het en 0.21 g.l-1 etanol oor dieselfde tydperk geproduseer het. Die rekombinante rasse wat in die studie gekonstrueer is, is 'n belangrike stap in die ontwikkeling van S. cerevisiae as KBP sellulolitiese gis. / The South African National Research Institute (SANERI) for financial support
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