Cell division of ggeneflesmus quadricauda (Turp.) Breb. (Scenedesmaceae) is enhanced by methylglyoxal, a general inhibitor of cell division, at threshold concentration in conjunction with treatment timing related to growth stage of batch cultures. At 0.5 mM methylglyoxal concentration, cell division was significantly enhanced in algae treated in the logarithmic phase. Specific growth rates of methylglyoxal-treated cultures were rapidly increased at the beginning of logarithmic phase. Cultures inoculated with high cell numbers were less sensitive, but still showed high specific growth rates in logarithmic phase. Cell division in cultures which had low cell numbers was inhibited by 0.5 mM methylglyoxal treatment. Both specific activity of Glyoxalase I and the ratio of Glyoxalase I to Glyoxalase II of methylgloxal-treated cultures were higher than those of controls (1.3 and 2.1- fold, respectively). Pyruvate concentration in treated cultures was increased after methylglyoxal treatment.

Trotz intensiver Forschung stellen bis heute maligne Tumorerkrankungen die zweithäufigste Todesursache in Industrienationen dar. Der Biochemiker Otto Warburg postulierte bereits vor 90 Jahren den nicht zu unterschätzenden Einfluss des Kohlenhydrat-Stoffwechsels in der malignen Entartung von Zellen. Als Nebenprodukt der Glykolyse entsteht das toxische Methylglyoxal, welches durch das nahezu ubiquitär vorkommende Glyoxalase-System neutralisiert werden kann. Speziell für die Glyoxalase 1 (Glo1) wird eine entscheidende Rolle in der Tumorgenese/-progression vermutet. In dieser Arbeit konnte basierend auf shRNA-vermittelten Glo1-knockdown Versuchen eine inhibitorische Rolle der Glo1 in der Ausbildung maligner Tumoreigenschaften wie Invasion, Migration und Proliferation in Astrozytomzellen gezeigt werden. Weiterhin wurde ein monoklonaler muriner Antikörper gegen die humane Isoform der Glo1 generiert und dessen Spezifität und Sensitivität bestätigt. Immunhistochemische Untersuchungen in primären Hirntumorgeweben offenbarten wiederum keine direkte Korrelation zwischen WHO-Stadium und Expressionsgrad bzw. subzellulärer Lokalisation der Glo1. Expressionsanalysen in malignen Tumorzelllinien unterschiedlicher Genese demonstrierten, dass besonders fortgeschrittene, therapieresistente Mamma- und Prostatakarzinomzellen niedrige Glo1-Level aufweisen. Basierend auf diesen Ergebnissen wurde ein zuvor identifizierter Inhibitor der Glo1 – das Phytotherapeutikum Curcumin – auf einen potentiellen Effekt in der Tumorprogression untersucht. Curcumin demonstrierte eine signifikante Reduktion der Proliferationsrate aller getesteter Tumorzelllinien. Darüber hinaus konnte ein in vivo -Tumormodell etabliert werden, mit welchem eine signifikante Tumorreduktion durch intraperitoneale und orale Applikation von Curcumin in Her-2/neu überexprimierenden Fibroblastenzellen erzielt wurde.

Given that chronic hyperglycemia generates toxic methylglyoxal, the detoxifying effect of glyoxalase-1 (Glo1) on chronic hyperglycemia induced explant-derived cardiac stem cell (EDC) dysfunction was investigated. Wildtype (WT) and Glo1 over-expressing (Glo1TG) mice with or without streptozotocin treatment were studied. Hyperglycemia reduced overall culture yields while increasing the reactive dicarbonyl content within WT mice. These intrinsic cell changes reduced the angiogenic potential and nanoparticle production by hyperglycemic EDCs while promoting cell senescence. Compared to transplant of normoglycemic WT EDCs, hyperglycemic EDCs reduced myocardial function following infarction by inhibiting angiogenesis and endogenous repair mechanisms. In contrast, EDCs from hyperglycemic Glo1TG mice decreased reactive dicarbonyl content and restored culture yields. Intramyocardial injection of hyperglycemic Glo1TG EDCs also boosted myocardial function and reduced scarring. These findings demonstrate that, while chronic hyperglycemia decreases the regenerative performance of EDCs, over-expression of Glo1 reduces dicarbonyl stress and rescues the adverse effects of hyperglycemia on EDCs.

Inflammation caused by oxidative stress (ROS) demonstrates an essential mechanism in the pathogenesis of acute pancreatitis (AP). Important sources for ROS comprise the reactive compound methylglyoxal (MGO) itself and the MGO-derived formation of advanced glycation end-products (AGEs). AGEs bind to the transmembrane receptor RAGE and activate NF-κB, and lead to the production of pro-inflammatory cytokines. MGO is detoxified by glyoxalase-I (Glo-I). The importance of Glo-I was shown in different models of inflammation and carcinogenesis. Nevertheless, the role of Glo-I and MGO in AP has not been evaluated so far. This study analyzed Glo-I in cerulein-(CN)-induced AP and determined the effects of Glo-I knockdown, overexpression and pharmacological modulation. Methods: AP was induced in C57BL6/J mice by i.p. injection of CN. Glo-I was analyzed in explanted pancreata by Western Blot, qRT-PCR and immunohistochemistry. AR42J cells were differentiated by dexamethasone and stimulated with 100 nM of CN. Cells were simultaneously treated with ethyl pyruvate (EP) or S-p-bromobenzylglutathione-cyclopentyl-diester (BrBz), two Glo-I modulators. Knockdown and overexpression of Glo-I was achieved by transient transfection with Glo-I siRNA and pEGFP-N1-Glo-I-Vector. Amylase secretion, TNF-α production (ELISA) and expression of Glo-I, RAGE and NF-κB were measured. Results: Glo-I was significantly upregulated on protein and mRNA levels in CN-treated mice and AR42J cells. Dexamethasone-induced differentiation of AR42J cells increased the expression of Glo-I and RAGE. Treatment of AR42J cells with CN and EP or BrBz resulted in a significant reduction of CN-induced amylase secretion, NF-κB, RAGE and TNF-α. Overexpression of Glo-I led to a significant reduction of CN-induced amylase levels, NF-κB expression and TNF-α, whereas Glo-I knockdown revealed only slight alterations. Measurements of specific Glo-I activity and MGO levels indicated a complex regulation in the model of CN-induced AP. Conclusion: Glo-I is overexpressed in a model of CN-induced AP. Pharmacological modulation and overexpression of Glo-I reduced amylase secretion and the release of pro-inflammatory cytokines in AP in vitro. Targeting Glo-I in AP seems to be an interesting approach for future in vivo studies of AP.

No description available.

Magister Scientiae - MSc (Biotechnology) / Soybean have been listed as a priority commodity crop in South Africa (SA) and provide a good source of protein to the population. Therefore, soybean has been earmarked as an important food security crop and strategies are currently being discussed at governmental level to increase and sustain soybean production. However, the SA landscape poses many challenges to the agricultural sector such as prolong drought periods, flooding, nutrient poor soils, saline soils and heavy metal contaminated soils. Heavy metal (HM) contamination is becoming a serious concern and is aggravated by historical mining in SA. Indeed, SA has established itself as the number one ranked mining country in the world and is frequently mining metals such as chromium, vanadium, gold, zirconium, platinum, and antimony. Prolong rainfall near mining areas leads to acid mine drainage which lowers the soil pH to approximately two. These highly acidic soils will solubilize the metals and cause the metals to leach into river systems as well as the water table leading to increase heavy metal contamination in nearby soil sites. This increase metal content negatively affects seed germination and overall plant development. Nonetheless, plants have evolved numerous internal mechanisms that help them to survive HM toxicity; by either avoiding or tolerating the stress. Two stress-activated pathways that help the plant tolerate stress have attracted much interest i.e. the glyoxalase system and reactive oxygen species (ROS) - antioxidant system as they detoxify methylglyoxal (MG) and ROS. / 2021-08-31

Enolate formation by proton abstraction from an sp3-hybridized carbon atom situated next to a carbonyl or carboxylate group is an abundant process in nature. Since the corresponding nonenzymatic process in water is slow and unfavorable due to high intrinsic free energy barriers and high substrate pKa s, enzymes catalyzing such reaction steps must overcome both kinetic and thermodynamic obstacles. Computer simulations were used to study enolate formation catalyzed by glyoxalase I (GlxI) and 3-oxo-Δ5-steroid isomerase (KSI). The results, which reproduce experimental kinetic data, indicate that for both enzymes the free energy barrier reduction originates mainly from the balancing of substrate and catalytic base pKas. This was found to be accomplished primarily by electrostatic interactions. The results also suggest that the remaining barrier reduction can be explained by the lower reorganization energy in the preorganized enzyme compared to the solution reaction. Moreover, it seems that quantum effects, arising from zero-point vibrations and proton tunnelling, do not contribute significantly to the barrier reduction in GlxI. For KSI, the formation of a low-barrier hydrogen bond between the enzyme and the enolate, which is suggested to stabilize the enolate, was investigated and found unlikely. The low pKa of the catalytic base in the nonpolar active site of KSI may possibly be explained by the presence of a water molecule not detected by experiments. The hemoglobin-degrading aspartic proteases plasmepsinI and plasmepsin II from Plasmodium falciparum have emerged as putative drug targets against malaria. A series of C2- symmetric compounds with a 1,2-dihydroxyethylene scaffold were investigated for plasmepsin affinity, using computer simulations and enzyme inhibition assays. The calculations correctly predicted the stereochemical preferences of the scaffold and the effect of chemical modifications. Calculated absolute binding free energies reproduced experimental data well. As these inhibitors have down to subnanomolar inhibition constants of the plasmepsins and no measurable affinity to human cathepsin D, they constitute promising lead compounds for further drug development.

One of the major outcomes of a genome sequencing project is the availability of amino acid sequences of all the proteins encoded in the genome of the organism concerned. However, most commonly, for a substantial proportion of the proteins encoded in the genome no information in function is available either from experimental studies or by inference on the basis of homology with a protein of known function. Even if the general function of a protein is known, the region of the protein corresponding to the function might be a domain and there may be additional regions of considerable length in the protein with no known function. In such cases the information on function is incomplete. Lack of understanding of the repertoire of functions of proteins encoded in the genome limits the utility of the genomic data. While there are many experimental approaches available for deciphering functions of proteins at the genomic scale, bioinformatics approaches form a good early step in obtaining clues about functions of proteins at the genomic scale (Koonin et al, 1998). One of the common bioinformatics approaches is recognition of function by homology (Bork et al, 1994). If the evolutionary relationship between two proteins, one with known function and the other with unknown function, could be established it raises the possibility of common function and 3-D structure for these proteins(Bork and Gibson, 1996). While this approach is effective its utility is limited by the ability of the bioinformatics approach to identify related proteins when their evolutionary divergence is high leading to low amino acid sequence similarity which is typical of two unrelated proteins (Bork and Koonin, 1998). Use of 3-D structural information, obtained by predictive methods such as fold recognition, has offered approaches towards increasing the sensitivity of remote homology detection 9e.g., Kelley et al, 2000; Shi et al, 2001; Gough et al, 2001). The work embodied in this thesis has the general objective of analysis of evolution of structural features and functions of families of proteins and design of new bioinformatics approaches for recognizing distantly related proteins and their applications. After an introductory chapter, a few chapters report analysis of functional and structural features of homologous protein domains. Further chapters report development and assessment of new remote homology detection approaches and applications to the proteins encoded in two protozoan organisms. A further chapter is presented on the analysis of proteins involved in methylglyoxal detoxification pathways in kinetoplastid organisms. Chapter I of the thesis presents a brief introduction, based on the information available in the literature, to protein structures, classification, methods for structure comparison, popular methods for remote homology detection and homology-based methods for function annotation. Chapter 2 describes the steps involved in the update and improvements made in this database. In addition to the update, the domain structural families are integrated with the homologous sequences from the sequence databases. Thus, every family in PALI is enriched with a substantial volume of sequence information from proteins with no known structural information. Chapter 3 reports investigations on the inter-relationships between sequence, structure and functions of closely-related homologous enzyme domain families. Chapter 4 describes the investigations on the unusual differences in the lengths of closely-related homologous protein domains, accommodation of additional lengths in protein 3-D structures and their functional implications. Chapter 5 reports the development and assessment of a new approach for remote homology detection using dynamic multiple profiles of homologous protein domain families. Chapter 6 describes development of another remote homology detection approach which are multiple, static profiles generated using the bonafide members of the family. A rigorous assessment of the approach and strategies for improving the detection of distant homologues using the multiple profile approach are discussed in this chapter. Chapter 7 describes results of searches made in the database of multiple family profiles (MulPSSM database) in order to recognize the functions of hypothetical proteins encoded in two parasitic protozoa. Chapter 8 describes the sequence and structural analyses of two glyoxalase pathway proteins from the kinetoplastid organism Leishmania donovani which causes Leishmaniases. An alternate enzyme, which would probably substitute the glyoxalase pathway enzymes in certain kinetoplastid organisms which lack the glyoxalase enzymes are also discussed. Chapter 9 summarises the important findings from the various analyses discussed in this thesis. Appendix describes an analysis on the correlation between a measure of hydrophobicity of amino acid residues aligned in a multiple sequence alignment and residue depth in 3-D structures of proteins.

Diabetes is a chronic disease with high prevalence and significant morbidity. Chronic changes in the wall of small and large vessels lead to main diabetes complications. Apart from long- term hyperglycemia, several factors are involved in the development of diabetes vasculopathy. The aim of this work was to describe new early biomarkers of these vascular changes, to identify risky patients. Alongside, gene polymorphisms involved in protective pathways of glucose metabolism were studied. In three human studies with Type 1 (T1D) and Type 2 (T2D) diabetes patients special biochemical parameters of receptor for advanced glycation endproducts (RAGE) and its ligands, deglycation enzyme glyoxalase 1 (GLO1) and fructosamine 3-kinase (FN3K) gene polymorphisms were analyzed. Non-invasive measurement of glycation by skin autofluorescence (SAF) was assessed in all subjects. Soluble RAGE, HMGB1 and endothelial dysfunction markers were increased in patients with diabetes as compared with controls, however the differences between T1D and T2D were not significant. For the first time, an association between FN3K (rs1056534) and (rs3848403) polymorphism and sRAGE concentration in diabetes was shown. GLO1 (rs4746) polymorphism was associated with changes in endothelial dysfunction. Patients with diabetes had higher...

Aquesta tesi demostra que el peroxinitrit produït com a conseqüència del pèptid beta-amiloide (A) contribueix l'augment de la relació A42/A40 que ocorre a la malaltia d'Alzheimer. L'A42 contribueix a l'aparició de la malaltia degut a la seva major toxicitat (quan es compara amb l'A40) que resulta d'una gran estabilitat i capacitat agregativa. A més el peroxinitrit incrementa la toxicitat d'aquest degut a què potencia la seva agregació en forma d'oligomers altament tòxics. De fet els oligomers formats de nitro-A42 presenten una major toxicitat que aquells formats de A42 . En conjunt aquest resultats senyalen l'important paper que l'A42 té en la malaltia d'Alzheimer. Per altra banda, des de la identificació dels agregats d'A i la subseqüent formació dels cabdells neurofibrilars (NFT) com a els dos trets distintius de la malaltia, un gran esforç s'ha dedicat a establir els mecanismes moleculars que uneixen ambdós processos. Aquesta tesi demostra que el peroxinitrit format a partir de l'agregació de d'Ai la conseqüent nitrotirosinació de proteïnes, potencia l'agregació de la proteïna tau en forma de fibres. D'aquesta forma, la nitrotirosinació de la proteïna triosafosfat isomerasa (TPI) podria ser el vincle entre la toxicitat derivada del agregats d'Ai la patologia derivada de la proteïna tau. Per tant, la nitrotirosinació de la TPI podria explicar la progressió temporal que ocorre als cervells de pacients amb la malaltia d'Alzheimer des de la toxicitat induïda per l'Ai l'aparició dels NFT. Els resultats presentats en aquesta tesi podrien obrir nous aspectes en la recerca de la malaltia d'Alzheimer així com en altres malalties que cursin amb estrès oxidatiu i plegament erroni de proteïnes. / This thesis demonstrates that amyloid ß-peptide (Aß)-induced peroxynitrite contributes to the switch of the Aβ42/Aβ40 ratio that occurs in Alzheimer's disease (AD). Since Aβ42 is more toxic due to its higher aggregation and stability, it contributes to the trigger of the disease. In addition the aggregation of Aβ42 in form of the highly toxic oligomers is incremented by the presence of peroxynitrite. Moreover, these nitro-Aß42 oligomers are more toxic than those non-nitrated. All these results support the important role of peroxynitrite in AD etiology. Furthermore, since the identification of Aß accumulation and the subsequent formation of neurofibrillary tangles (NFT) as the two defining pathological hallmarks of AD, a fair amount of research on AD has been driven by the need to find the molecular mechanism linking Aß and NFT. This thesis shows the Aß-induced peroxynitrite, and the consequent nitrotyrosination of proteins, promotes tau fibrillization. Thus triosephosphate isomerase (TPI) nitrotyrosination could be the link between Aß-induced toxicity and tau pathology. Therefore, TPI nitrotyrosination may explain the temporal progression from Aß toxicity to NFT formation in AD brain. The work presented in this thesis could open a novel angle in the research of the pathophysiology of AD and could also have an impact to the research in other neurodegenerative diseases involving oxidative stress and protein misfolding.