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Intracellular hexokinase localization in hybridoma cultures implications for regulation of metabolism and cell death /Clark, Lindsey M. January 1900 (has links)
Thesis (Ph. D. in Chemical Engineering)--Vanderbilt University, Aug. 2005. / Title from title screen. Includes bibliographical references.
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Molekulare Zielstrukturen im Alloxan-induzierten Diabetesmodell der MausSchulte im Walde, Sabine 17 December 2004 (has links) (PDF)
Alloxan (ALX) ist ein klassisches Diabetogen, welches in Nagetieren spezifisch pankreatische ß-Zellen zerstört und Symptome induziert, die dem humanen Typ-1-Diabetes vergleichbar sind. Durch eine einmalige, intravenöse (iv) Injektion einer subtoxischen Dosis von 50 mg ALX/kg Körpergewicht (KG) werden Schäden an der ß-Zelle hervorgerufen, die innerhalb von 48-72 Stunden in 50% der Mäuse einen Diabetes auslösen (Schwellenwert Euglykämie zu Hyperglykämie ist 11,1 mmol/l). Das toxische Potential von ALX besteht in der Generierung von reaktiven Sauerstoffspezies (ROS), vorwiegend Superoxidanion-, Wasserstoffperoxid- und Hydroxylradikalen. Ziel der vorliegenden Arbeit war zu untersuchen, ob durch ALX präferentiell Strukturen der ß-Zelle zerstört werden, die essentiell für die ß-Zellfunktion – die Insulinproduktion – sind. Diese sind u.a. der Glucosetransporter 2 (GLUT2), die Glucokinase und das Proinsulin. Daran anschliessend stellte sich die Frage, ob ALX-Toxizität durch Vorbehandlung mit D-Glucose (D-G), 5-Thio-D-Glucose (5-T-G) oder mit Zink-angereichertem Trinkwasser – zur Anreicherung des Antioxidants Metallothionein - verhindert werden kann. Hierzu wurden männliche C57BL/6- und 129S3-Mäuse entweder einmalig iv mit D-G oder 5-T-G vorbehandelt oder die Mäuse erhielten eine Woche vor der ALX-Injektion und über die gesamte Versuchsdauer hinweg Zink-angereichertes Trinkwasser. Anschliessend wurde der Einfluss auf den ALX-induzierten Diabetes, die orale Glucosetoleranz und die mRNA-Expression der oben genannten Gene mittels RT-PCR untersucht. Der Gesamtinsulingehalt der ALX-behandelten Pancreata wurde über die Bestimmung des immunreaktiven Insulins ermittelt. In der vorliegenden Arbeit wird gezeigt, dass 1.) die Vorbehandlung mit D-G den ALX-induzierten Diabetes signifikant (p0,001) verhinderte; 2.) trotz Euglykämie in mit D-G- und ALX-behandelten Mäusen eine pathologische orale Glucosetoleranz über Wochen als ALX-Folgeschaden persistierte; 3.) die Vorbehandlung mit 5-T-G, dem chemisch der D-G ähnlichsten Analog, jedoch den ALX-induzierten Diabetes signifikant (p0,001) potenzierte; 4.) Zink-angereichertes Trinkwasser die ALX-induzierte Hyperglykämie signifikant (p0,001) reduzierte; 5.) ALX zunächst die mRNA-Expression des GLUT2 signifikant (p0,001) reduzierte und nachfolgend auch signifikant (p0,05) die mRNA-Expression der Glucokinase, wenn auch weniger ausgeprägt als für die GLUT2-Expression; 6.) ALX keine Veränderung der mRNA-Expression von Proinsulin auslöste; 7.) die Vorbehandlung mit D-G signifikant (p0,05) die ALX-induzierte Reduktion der mRNA-Expression von GLUT2 und der Glucokinase verhinderte und 8.) der Insulingehalt im gesamten Pankreas 24 h nach ALX-Injektion signifikant (p0,05) reduziert wurde. Es wird geschlussfolgert, dass der GLUT2 und die Glucokinase primäre Zielstrukturen der ALX-Toxizität unter den verwendeten Versuchsbedingungen sind. Diese Läsionen sind die Ursache für den Diabetes. Durch Vorbehandlung mit D-G können der GLUT2 und die Glucokinase vor ALX-Toxizität geschützt werden, obwohl trotz Euglykämie unter physiologischen Bedingungen eine pathologische orale Glucosetoleranz als ALX-Folgeschaden in den Mäusen persistierte. Es muß noch geklärt werden, worin die Gründe für den protektiven Effekt der D-G und den potenzierenden Effekt der 5-T-G liegen und inwieweit ALX-induzierte Radikale selektiv wirksam sind, oder ob diese Selektivität auf anderen Mechanismen, wie z.B. Transkriptionsfaktoren, beruht. Letztendlich zeigen diese Befunde, dass sich der pathogenetische Mechanismus von ALX von anderen Diabetogenen unterscheidet, wie z.B. Streptozotozin, welches selektiv den GLUT2 schädigt, der durch Vorbehandlung mit 5-T-G vor der Streptozotocin-Toxizität geschützt werden kann. Daraus ist abzuleiten, dass es in der Präventivmedizin unterschiedlicher Vorsorgemassnahmen bedarf, um Risiko-patienten vor der Manifestation eines Diabetes mellitus zu schützen. / Type 1 diabetes results from irreversible damage of insulin-producing ß-cells. In laboratory animals, diabetes can be induced with alloxan (ALX). ALX is a potent generator of reactive oxygen species (ROS), which can mediate ß-cell toxicity. However, the initial lesions on essential ß-cell structures are not known. In this study, we analyzed the effect of ALX on the glucose transporter 2 (GLUT2), glucokinase and proinsulin. For this purpose, we investigated the effect of pretreatment with the glucose analogues D-glucose (D-G) and 5-thio-D-glucose (5-T-G), as well as with zinc-enriched drinking water to induce the antioxidant metallothionein, on ALX-induced diabetes, on oral glucose tolerance (OGT) and on the mRNA-expression of the above mentioned genes with semiquantitative RT-PCR in male C57BL/6 and 129S3 mice. The total insulin content of ALX-treated pancreata was determined as immune reactive insulin. One single intravenous (iv) injection of 50 mg ALX/kg body weight (bwt) induced diabetes in 50% of mice of both strains (blood glucose level 11.1 mmol/l). One single iv preinjection of D-G prevented significantly (p0,001) diabetes in both strains, yet, in these euglycemic mice, an impaired oral glucose tolerance persisted. In contrast, the pretreatment with a single injection of 5-T-G potentiated significantly (p0,001) the toxicity of ALX. Administration of zinc-enriched drinking water, however, reduced ALX-induced hyperglycemia (p0,001). The mRNA-expression of GLUT2 and glucokinase was time-dependently reduced and the effect was more pronounced for GLUT2 (p0,001) than for glucokinase (p0,05). The pretreatment with D-G protected against the mRNA-reduction of both GLUT2 and glucokinase (p0,05). Interestingly, the mRNA-expression of proinsulin remained unaffected as well as the pancreatic total insulin content. A significant (p0,05) reduction of pancreatic insulin content was found after 24 h. In conclusion, ALX exerts differential toxicity on essential ß-cell structures. This toxic effect was more pronounced for GLUT2 than for glucokinase mRNA. Pretreatment with D-G prevented ALX-toxicity, whereas in euglycemic mice an impaired oral glucose tolerance persisted. It has to be elucidated, whether ALX-induced ROS select essential ß-cell structures or whether, as one possibility, transcription factors in the ß-cell are specifically directing ROS to GLUT2 and glucokinase mRNA. Finally, these results differ from those obtained with other diabetogens, e.g., streptozotocin, which exerts selective toxicity on the GLUT2 and which is prevented by 5-T-G. However, diabetogens damage ß-cell function through different pathogenic pathways and, therefore, different interventional regimen may be required to prevent type 1 diabetes in individuals at risk.
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Comparative study of the effect of silver nanoparticles on the hexokinase activity from human and Trypanosoma bruceiMlozen, Madalitso Martin January 2015 (has links)
No description available.
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Elucidating enzyme catalytic power and protein-ligand dynamics of human glucokinase: the role of modern allosteryLi, Quinn 01 July 2018 (has links)
Glucokinase (GK) is an enzyme that catalyzes the ATP-dependent phosphorylation of glucose to form glucose-6-phosphate, and it is a tightly regulated checkpoint in glucose homeostasis. The monomeric enzyme possesses a highly exotic kinetic profile, with a sigmoidal dependence on glucose, which has been the source of vigorous investigation and debate in the last several decades. This unique regulatory behavior can be thought of as a remarkable glucose sensor, which may result in hyperglycemia when it is not active enough and hypoglycemia when it is too active. This interdisciplinary study, which draws on small angle X-ray scattering (SAXS) integrated with atomistic molecular dynamics simulations and experimental glucose binding thermodynamics, I reveal the critical regulation of the glucose sensor is due to a solvent controlled switch. Moreover, this solvent controlled switch manifests a regulatory mechanism of GK; a specific local conformational change that leads to an enzyme structure that has a much more favorable solvation energy than most of the protein ensemble. These findings have direct implications for the design of small molecule GK activators as anti- diabetes therapeutics as well as for understanding how proteins can be designed to have built-in regulatory functions via solvation energy dynamics.
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Regulation of rat Liver Glucokinase Gene Expression by Sterol Regulatory Element Binding Protein-1a and Forkhead box classO1 Transcription factorsGanjam, Goutham Kumar 01 November 2007 (has links)
No description available.
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The role of genetic variation in glucokinase and glucokinase regulatory protein in diabetes and related traitsBeer, Nicola L. January 2011 (has links)
The rising prevalence of type 2 diabetes (T2D) is a global problem, and suggests that we need better therapeutic strategies against this disease. The glycolytic enzyme glucokinase (GCK) catalyses the phosphorylation of glucose, and is a well-established T2D drug target. Rare GCK mutations cause monogenic beta-cell dysfunction, whilst common genetic variants within GCK are associated with fasting plasma glucose (FPG) levels and T2D risk. Since GCK is expressed in both the pancreas and liver, pharmacological GCK activation provides the promise of a two-pronged attack on hyperglycaemia. In vivo, GCK activity is modulated by the hepatic inhibitor glucokinase regulatory protein (GKRP, gene GCKR). GKRP negatively regulates GCK activity competitively with respect to glucose, and is controlled by fructose 6- and fructose 1-phosphate (F6P and F1P), which compete with each other for binding and enhance or diminish GCK inhibition respectively. GKRP also sequesters GCK in the nucleus and paradoxically stabilises the enzyme. As GCK and its regulatory protein are fundamental to glucose homeostasis, we aimed to investigate the role of genetic variation in both GCK and GCKR to further our understanding of these important T2D drug targets in a system that would be relevant to man. I demonstrated that two novel GCK mutations (T103S and V389L) identified in patients with hyperinsulinaemic hypoglycaemia were kinetically activating and through structural modelling identified a novel regulatory site for GCK activation by small molecular activators. Genome-wide association studies (GWAS) identified GCKR as a regulator of FPG and triglyceride levels, and showed a role for GKRP in T2D risk. Unlike most GWAS hits, this signal included a non-synonymous variant within GCKR (P446L), thus facilitating functional studies. P446L-GKRP was characterised kinetically and at the cellular sequestration-level. This variant showed diminished F6P-mediated modulation, which was proposed to reduce hepatic GCK inhibition, increase glycolytic flux (decreasing FPG), and feed metabolites into liver pathways (elevating triglycerides). As GCKR was not expressed at functional levels within human islets, this phenotype was thought to be driven by the liver. Preliminary analysis at the cellular level was inconclusive, with optimisation required to study human P446L-GKRP in this cellular system. Finally, I showed that mutations within GCKR are not a common cause of “GCK-Like” phenotypes in man, despite the regulatory protein directly modulating GCK activity. These data provide further insight as to the pathogenic consequences of perturbing GCK activity. This must be considered if this enzyme is to be the subject of therapeutic intervention in T2D.
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Kinetic modelling of wine fermentations : why does yeast prefer glucose to fructoseMocke, Leanie 03 1900 (has links)
Thesis (MSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: In the present-day competitive global market, wine industries are constantly
aiming to improve the wine-making process,including the role of yeast. The
most commonly used wine yeast is Saccharomyces cerevisiae, which is able to
produce high quality wines, but problem fermentations do sometimes arise.
The occurrence of stuck and sluggish fermentations pose a serious problem
leading to loss of productivity and quality. Although the precise mechanism
leading to stuck fermentations is unknown, they are often correlated with
high fructose to glucose ratios in the wine-must. S. cerevisiae is a glucophylic
yeast, indicating its preference for consuming glucose over fructose. Both these
hexose sugars are present in unfermented wine must, mostly in equal concentrations.
As fermentation progresses, glucose is consumed at a faster rate than
fructose, leading to an increase in the fructose to glucose ratio. Yeast are left
with the undesirable fructose at the later stages of fermentation, when the
environmental stresses on the yeast can lead to stuck or sluggish fermentation.
This residual fructose can lead to undesirable sweetness, as fructose is
about twice as sweet as glucose. Even with the extensive research into yeast
metabolism, there is as yet no definitive explanation as to why yeasts ferment
glucose faster than fructose.
This study aimed to investigate the mechanism responsible for the faster consumption
of glucose over fructose of a commercially used wine yeast strain
S. cerevisiae VIN 13. The first two steps of sugar metabolism, uptake and
phosphorylation, were investigated as the possible sites of discrepancy in fermentation
rates. Enzyme rates and affinities for both glucose and fructose as substrates for the relevant enzymes were experimentally determined. These
kinetic parameter values were used to improve an existing model of yeast glycolytic
pathway to model wine fermentations. The feasibility of constructing
and validating a kinetic model of wine fermentations were investigated, by
comparing model predicted fluxes with experimentally determined fluxes.
Another aspect of this study was an investigation into the effect of hexose
sugar type on fermentation profiles. Wine fermentations were done with only
one hexose sugar as carbon source to determine if it has an effect on the flux
through metabolism.
This work succeeded in the construction of a kinetic model that distinguished
between glucose and fructose as carbon source. The glucose was consumed
faster than fructose, with control lying in the hexose transport step. It was
also established that fermentation prfiles of fermentations with only one sugar
was the same for both one sugar type fermentations. Fermentation with either
glucose or fructose as the sole carbohydrate source had the same specfic
production and consumption rates as normal fermentations with both sugars.
Construction of detailed kinetic models can aid in the metabolic and cellular
engineering of novel yeast strains. By identifying the importance of hexose
transport, and thus the glucophilic character of the yeast, in flux control, yeast
transporters can be targeted for strain improvement. This may in turn lead to
more effective fermentation practices for controlling problem fermentations, or
to the development of novel strains that utilizes fructose in the same manner as
glucose, and in so doing lower the risk of stuck or sluggish wine fermentation. / AFRIKAANSE OPSOMMING: In die hedendaagse kompeterende wynmark is wynmakers aanhoudend besig
om die wynmaak proses te verbeter en dit sluit die verbetering van wyngis
in. Die mees algemeenste gebruikte wyngis is Saccharomyces cerevisiae, omdat
dit wyn van gehalte produseer, maar probleem fermentasies kom wel voor.
Die verskynsel van vasval of stadige fermentasies kan lei tot die verlies van
produksie en kwaliteit. Die oorsaak van probleem fermentasies is gewoontlik
veelvoudig, maar die verhouding van glukose tot fruktose in die wyn-mos kan
ongunstig raak om fermentasies te onderhou. S. cerevisiae is 'n glukofiliese
gis, wat sy voorkeur om glukose bo fruktose te gebruik beskryf. Albei hierdie
heksose suikers is teenwoordig in ongefermenteerde wyn-mos, meestal in gelyke
hoeveelhede. Soos fermentasies vorder word glukose vinniger verbruik as
fruktose wat lei tot 'n toename in die fruktose tot glukose verhouding. Die gis
moet dus die fruktose in die later stadium van fermentasie gebruik wanneer
die omgewings druk op die gis kan lei tot probleem fermentasies. Die oorblywende
fruktose kan lei tot ongewenste soetheid aangesien fruktose twee keer
soeter is as glukose. Selfs met die ekstensiewe navorsing met betrekking tot
gis metabolisme is daar nog nie 'n verduideliking hoekom gis glukose vinniger
as fruktose gebruik nie.
Hierdie studie het beoog om die meganisme wat lei tot die vinniger verbruik
van glukose oor fruktose te ondersoek vir 'n kommersieël gebruikte gis S. cerevisiae VIN 13. Die eerste twee stappe van suiker metabolisme, suiker opname
en fosforilasie, was ondersoek as die moontlike punt van die verskil in fermentasie
tempo. Ensiem snelhede en affiniteite vir beide glukose en fruktose as substrate vir die ensieme van belang was eksperimenteel bepaal. Hierdie
waardes is gebruik om 'n bestaande model van gis glikolise aan te pas vir wyn
fermentasies. Die uitvoerbaarheid van saamstel en valideer van 'n kinetiese
model van wyn fermentasies was ondersoek, deur model voorspelde fluksie
waardes met eksperimentele fluksie waardes te vergelyk.
'n Ander aspek van die studie was die ondersoek van die effek van heksose
suiker tipe op fermentasie profiel. Wyn fermentasies is gedoen met slegs een
heksose suiker as koolstof bron om te bepaal of dit 'n invloed het op die fluksie
deur metabolisme.
Hierdie werk het daarin geslaag om 'n kinetiese model saamtestel wat onderskei
tussen glukose en fruktose as koolstof bron. Die glukose is vinniger verbruik as
fruktose, met beheer gesetel in die heksose opname stap. Dit was ook vasgestel
dat fermentasie profiele van fermentasies met slegs een suiker nie verskil het
vir fermentasies met slegs fruktose of glukose. Fermentasies met slegs een
suiker het dieselfde spesifieke produksie en konsumpsie tempo gehad as die
normale fermentasie met albei suikers. Die konstruksie van 'n gedetailleerde
kinetiese model kan gebruik word in die metaboliese en sellulêre ontwikkeling
van nuwe gisstamme. Deur die ontdekking van die belangrikheid van heksose
opname in fluksie beheer, wat lei tot die glukofiliese karakter van gis, kan gis
opname geteiken word vir gis ontwikkeling. Dit mag om die beurt lei tot meer
effektiewe fermentasie praktyk in die beheer van probleem fermentasies, of die
ontwikkeling van nuwe stamme wat fruktose in dieselfde manier as glukose
benut, en sodoende die risiko van vasval of stadige wyn fermentasies verlaag. / National Research Foundation / Post-graduate Merit Bursary
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Purification And Characterization Of Hexokinase Isoenzymes From Rhizopus OryzaeDedeoglu, Didem 01 April 2005 (has links) (PDF)
ABSTRACT
PURIFICATION AND CHARACTERIZATION OF HEXOKINASE ISOENZYMES FROM Rhizopus oryzae
Dedeoglu, Didem
MS., Department of Biotechnology
Supervisor: Prof.Dr. Haluk Hamamci
Co-supervisor: Dr. Seyda Aç / ar
February 2007, 116 pages
Glycolysis is the central metabolic pathway for living organisms. Its regulation is important for the yield of the end products which are industrially important. These end products, like lactic acid produced by Rhizopus oryzae, are industrially important.
Rhizopus oryzae is a filamentous fungus producing lactic acid and ethanol. The lactic acid yield of R. oryzae is low (& / #61566 / 70 %) compared to that of lactic acid bacteria (& / #61502 / 95 %) still it is noteworthy because R. oryzae produces only the L (+) form of lactic acid which can be metabolized in the human body. The yield of an industrial process should be high for the feasibility of the production of a particular product. If a way can be found increase the flux through the glycolysis the yield of lactic acid may increase as well.
Keeping this in mind we wanted to focus on the first step of glycolysis, hexokinase of R. oryzae. Hexokinase catalyzes the reaction that converts glucose to glucose-6-phosphate. In this study for the first time the two isoenzymes of hexokinase of R. oryzae were purified and characterized by biochemically and kinetically
Hexokinase has two isoenzymes. The purified enzymes (isoenzymes1 & / isoenzymes2) obeyed Michealis-Menten Kinetics. The Km value of purified isoenzyme 1 is 0.16 mM and isoenzyme 2, 0.21 mM at pH 7.70 for glucose. The Km value of isoenzyme1 for fructose was 28.8 mM. Essentially isoenzyme 2 can not utilize fructose. None of the isoenzymes were inhibited by trehalose-6-phophate.The monomer moleculer weight of isoenzymes were estimated SDS PAGE analysis. There were two different values for molecular weight of isoenzmye 1 / 62.9 and 42.5 kDa and two values for isoenzyme 2 / 56.2 and 41.6 kDa
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Molekulare Zielstrukturen im Alloxan-induzierten Diabetesmodell der MausSchulte im Walde, Sabine 01 March 2004 (has links)
Alloxan (ALX) ist ein klassisches Diabetogen, welches in Nagetieren spezifisch pankreatische ß-Zellen zerstört und Symptome induziert, die dem humanen Typ-1-Diabetes vergleichbar sind. Durch eine einmalige, intravenöse (iv) Injektion einer subtoxischen Dosis von 50 mg ALX/kg Körpergewicht (KG) werden Schäden an der ß-Zelle hervorgerufen, die innerhalb von 48-72 Stunden in 50% der Mäuse einen Diabetes auslösen (Schwellenwert Euglykämie zu Hyperglykämie ist 11,1 mmol/l). Das toxische Potential von ALX besteht in der Generierung von reaktiven Sauerstoffspezies (ROS), vorwiegend Superoxidanion-, Wasserstoffperoxid- und Hydroxylradikalen. Ziel der vorliegenden Arbeit war zu untersuchen, ob durch ALX präferentiell Strukturen der ß-Zelle zerstört werden, die essentiell für die ß-Zellfunktion – die Insulinproduktion – sind. Diese sind u.a. der Glucosetransporter 2 (GLUT2), die Glucokinase und das Proinsulin. Daran anschliessend stellte sich die Frage, ob ALX-Toxizität durch Vorbehandlung mit D-Glucose (D-G), 5-Thio-D-Glucose (5-T-G) oder mit Zink-angereichertem Trinkwasser – zur Anreicherung des Antioxidants Metallothionein - verhindert werden kann. Hierzu wurden männliche C57BL/6- und 129S3-Mäuse entweder einmalig iv mit D-G oder 5-T-G vorbehandelt oder die Mäuse erhielten eine Woche vor der ALX-Injektion und über die gesamte Versuchsdauer hinweg Zink-angereichertes Trinkwasser. Anschliessend wurde der Einfluss auf den ALX-induzierten Diabetes, die orale Glucosetoleranz und die mRNA-Expression der oben genannten Gene mittels RT-PCR untersucht. Der Gesamtinsulingehalt der ALX-behandelten Pancreata wurde über die Bestimmung des immunreaktiven Insulins ermittelt. In der vorliegenden Arbeit wird gezeigt, dass 1.) die Vorbehandlung mit D-G den ALX-induzierten Diabetes signifikant (p0,001) verhinderte; 2.) trotz Euglykämie in mit D-G- und ALX-behandelten Mäusen eine pathologische orale Glucosetoleranz über Wochen als ALX-Folgeschaden persistierte; 3.) die Vorbehandlung mit 5-T-G, dem chemisch der D-G ähnlichsten Analog, jedoch den ALX-induzierten Diabetes signifikant (p0,001) potenzierte; 4.) Zink-angereichertes Trinkwasser die ALX-induzierte Hyperglykämie signifikant (p0,001) reduzierte; 5.) ALX zunächst die mRNA-Expression des GLUT2 signifikant (p0,001) reduzierte und nachfolgend auch signifikant (p0,05) die mRNA-Expression der Glucokinase, wenn auch weniger ausgeprägt als für die GLUT2-Expression; 6.) ALX keine Veränderung der mRNA-Expression von Proinsulin auslöste; 7.) die Vorbehandlung mit D-G signifikant (p0,05) die ALX-induzierte Reduktion der mRNA-Expression von GLUT2 und der Glucokinase verhinderte und 8.) der Insulingehalt im gesamten Pankreas 24 h nach ALX-Injektion signifikant (p0,05) reduziert wurde. Es wird geschlussfolgert, dass der GLUT2 und die Glucokinase primäre Zielstrukturen der ALX-Toxizität unter den verwendeten Versuchsbedingungen sind. Diese Läsionen sind die Ursache für den Diabetes. Durch Vorbehandlung mit D-G können der GLUT2 und die Glucokinase vor ALX-Toxizität geschützt werden, obwohl trotz Euglykämie unter physiologischen Bedingungen eine pathologische orale Glucosetoleranz als ALX-Folgeschaden in den Mäusen persistierte. Es muß noch geklärt werden, worin die Gründe für den protektiven Effekt der D-G und den potenzierenden Effekt der 5-T-G liegen und inwieweit ALX-induzierte Radikale selektiv wirksam sind, oder ob diese Selektivität auf anderen Mechanismen, wie z.B. Transkriptionsfaktoren, beruht. Letztendlich zeigen diese Befunde, dass sich der pathogenetische Mechanismus von ALX von anderen Diabetogenen unterscheidet, wie z.B. Streptozotozin, welches selektiv den GLUT2 schädigt, der durch Vorbehandlung mit 5-T-G vor der Streptozotocin-Toxizität geschützt werden kann. Daraus ist abzuleiten, dass es in der Präventivmedizin unterschiedlicher Vorsorgemassnahmen bedarf, um Risiko-patienten vor der Manifestation eines Diabetes mellitus zu schützen. / Type 1 diabetes results from irreversible damage of insulin-producing ß-cells. In laboratory animals, diabetes can be induced with alloxan (ALX). ALX is a potent generator of reactive oxygen species (ROS), which can mediate ß-cell toxicity. However, the initial lesions on essential ß-cell structures are not known. In this study, we analyzed the effect of ALX on the glucose transporter 2 (GLUT2), glucokinase and proinsulin. For this purpose, we investigated the effect of pretreatment with the glucose analogues D-glucose (D-G) and 5-thio-D-glucose (5-T-G), as well as with zinc-enriched drinking water to induce the antioxidant metallothionein, on ALX-induced diabetes, on oral glucose tolerance (OGT) and on the mRNA-expression of the above mentioned genes with semiquantitative RT-PCR in male C57BL/6 and 129S3 mice. The total insulin content of ALX-treated pancreata was determined as immune reactive insulin. One single intravenous (iv) injection of 50 mg ALX/kg body weight (bwt) induced diabetes in 50% of mice of both strains (blood glucose level 11.1 mmol/l). One single iv preinjection of D-G prevented significantly (p0,001) diabetes in both strains, yet, in these euglycemic mice, an impaired oral glucose tolerance persisted. In contrast, the pretreatment with a single injection of 5-T-G potentiated significantly (p0,001) the toxicity of ALX. Administration of zinc-enriched drinking water, however, reduced ALX-induced hyperglycemia (p0,001). The mRNA-expression of GLUT2 and glucokinase was time-dependently reduced and the effect was more pronounced for GLUT2 (p0,001) than for glucokinase (p0,05). The pretreatment with D-G protected against the mRNA-reduction of both GLUT2 and glucokinase (p0,05). Interestingly, the mRNA-expression of proinsulin remained unaffected as well as the pancreatic total insulin content. A significant (p0,05) reduction of pancreatic insulin content was found after 24 h. In conclusion, ALX exerts differential toxicity on essential ß-cell structures. This toxic effect was more pronounced for GLUT2 than for glucokinase mRNA. Pretreatment with D-G prevented ALX-toxicity, whereas in euglycemic mice an impaired oral glucose tolerance persisted. It has to be elucidated, whether ALX-induced ROS select essential ß-cell structures or whether, as one possibility, transcription factors in the ß-cell are specifically directing ROS to GLUT2 and glucokinase mRNA. Finally, these results differ from those obtained with other diabetogens, e.g., streptozotocin, which exerts selective toxicity on the GLUT2 and which is prevented by 5-T-G. However, diabetogens damage ß-cell function through different pathogenic pathways and, therefore, different interventional regimen may be required to prevent type 1 diabetes in individuals at risk.
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High-Yield Cellulosic Hydrogen Production by Cell-Free Synthetic Cascade Enzymes: Minimal Bacterial Cellulase Cocktail and Thermostable Polyphosphate GlucokinaseLiao, Hehuan 09 June 2011 (has links)
Hydrogen production from abundant renewable biomass would decrease reliance on crude oils, achieve nearly zero net greenhouse gas emissions, create more jobs, and enhance national energy security. Cell-free synthetic pathway biotransformation (SyPaB) is the implementation of complicated chemical reaction by the in vitro assembly of numerous enzymes and coenzymes. Two of the biggest challenges for its commercialization are: effective release of fermentable sugars from pretreated biomass, and preparations of thermostable enzymes with low-cost.
The hydrolysis performance of 21 reconstituted bacterial cellulase mixtures containing the glycoside hydrolase family 5 Bacillus subtilis endoglucanase, family 9 Clostridium phytofermentans processive endoglucanase, and family 48 Clostridium phytofermentans cellobiohydrolase was investigated on microcrystalline cellulose (Avicel) and regenerated amorphous cellulose (RAC). The optimal ratios for maximum cellulose digestibility were dynamic for Avicel but nearly fixed for RAC. Processive endoglucanase CpCel9 was most important for high cellulose digestibility regardless of substrate type. These results suggested that the hydrolysis performance of reconstituted cellulase cocktail strongly depended on experimental conditions.
Thermobifida fusca YX was hypothesized to have a thermophilic polyphosphate glucokinase. T. fusca YX ORF Tfu_1811 encoding a putative PPGK was cloned and the recombinant protein fused with a family 3 cellulose-binding module (CBM-PPGK) was over expressed in Escherichia coli. By a simple one-step immobilization, the half-life time increased to 2 h, at 50 °C. These results suggest that this enzyme was the most thermostable PPGK reported.
My studies would provide important information for the on-going project: high-yield hydrogen production from cellulose by cell-free synthetic enzymatic pathway. / Master of Science
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