The novel protein Carom is a homolog of the Drosophila protein, Nervous Wreck (Nwk). Carom was cloned as a homocysteine response protein in primary endothelial cells (EC). In order to discern the functionality of the protein in higher order mammals and potential role in homocysteine-inhibited angiogenesis, we performed basic characterization studies on Carom. Theoretical modeling of Carom matched the solved structure of Nwk. Using a lab-generated antibody against Carom’s F-BAR domain, it is evident that Carom is localized to the mitochondria and speckling in the nucleus of primary ECs. In order to perform biochemical and structural studies in primary ECs, Carom was cloned from an adenoviral shuttle vector to an adeno-associated virus (AAV) transfer vector. We created a multi-cistronic open reading frame with an N-terminal Flag and a cleavable C-terminal green fluorescent protein (mClover3). Primary ECs are difficult to transfect, so we optimized an AAV packaging system in order to get high titers and high purity AAV that can transduce our primary ECs. Carom is composed of several functional domains with the capability of binding to cell membranes and act as a scaffolding for attaching adaptor proteins. To isolate which domains are important to the partner binding and cellular localization, we serially truncated the domains from Carom starting from both the C-terminus and N-terminus. We demonstrated that the C-terminal region features some post-translational modifications creating the second band in western blots with lower mobility. Also, the F-BAR domain is responsible for translocalization of Carom from the cytoplasm to the cell membrane and nucleus. A novel mechanism is proposed for why Carom is upregulated in response to homocysteine (Hcy), an independent risk factor for cardiovascular disease. It is previously known that Hcy inhibits angiogenesis. Our data mining studies identified a potentially important receptor for angiogenesis in ECs, VEGFR2, being endocytosed and ultimately degraded. Through biotinylation assays, we determined that Carom does help enhance the endocytosis of VEGFR2 potentially leading to degradation via the lysosome. In summary, Carom is endogenously localized to the mitochondria in primary ECs, the C-terminus is post-translationally modified, the bipartite nuclear localization signal containing F-BAR domain localizes to the cell membrane and nucleus, and Carom enhances the endocytosis of VEGFR2. / Biomedical Sciences

Methionine synthases catalyze methyl transfer from 5-methyl-tetrahydrofolate (5-methyl-THF) to L-homocysteine (Hcy) in order to generate methionine (Met). Mammals, including humans, use a cobalamin dependent form, while fungi use a cobalamin independent protein called Met6p. The large structural differences between them make Met6p a potential anti-fungal drug target. Met6p is a 90 kDa protein with the active site located between two (βα)₈ barrels. The active site has a catalytic Zn²+ and binding sites for the two substrates, Hcy and folate. I present the crystal structures of three engineered variants of the Met6p enzyme from Candida albicans. I also solved Met6p in complex with several substrate and product analogs, including Hcy, Met, Gln, 5-methyl-THF-Glu₃ and Methotrexate-Glu₃ (MTX-Glu₃), and the bi-dentate ligand S-adenosyl homocysteine. Also described is a new fluorescence-based activity assay monitoring Hcy. Lastly, a high-throughput Differential Scanning Fluorimetry (DSF) assay was used to screen thousands of compounds in order to identify ligands which bind Met6p. My work details the mode of interaction of Hcy and folate with the Met6p protein. Several residues important to activity were discovered, like Asn 126 and Tyr 660, and proven to be important by site directed mutagenesis. Structural analysis revealed an important aspect of the mechanism. When Hcy binds to its pocket it makes strong ion pairs with the enzyme. In particular, 614 moves toward the substrate amine and triggers a rearrangement of active site loops; this draws the catalytic Zn²+ toward the Hcy thiol where a new ligand bond is formed, activating the thiol for methyl transfer. The work presented here lays the groundwork for structure based drug design and makes the development of Met6p specific bi-dentate ligands feasible. The fluorescence based activity assay I developed was successfully used to test the folate analog MTX-Glu₃, which inhibits with an IC₅₀ of ~4 mM. I also discovered our first bi-dentate ligand in the form of S-adenosyl homocysteine. / text

Thesis (MMedSc)--Stellenbosch University, 2013. / ENGLISH ABSTRACT: The aetiology of multiple sclerosis (MS) remains largely unknown due to the multifactorial nature of disease susceptibility determined by both environmental and genetic factors. Progress has been made in identifying the genetic component of MS , as well as the possible interactions with the environment. In this study single nucleotide polymorphisms (SNPs) in the FTO (rs9939609, Intron 1 T>A), MTR (rs1805087, 2756 A>G), MTRR (rs1801394, 66 A>G), MTHFR (rs1801133, 677 C>T and rs1801131, 1298 A>C) and COMT (rs4680, 472 G>A) genes involved in the methylation metabolic pathway were studied in the context of MS. The overall objective of this study was to elucidate the mechanism underlying raised homocysteine levels in MS patients. The specific aims were 1) to analytically validate high throughput real-time polymerase chain reaction (RT-PCR) genotyping assays for the 6 selected SNPs against direct sequencing as the gold standard for 2) possible integration into a pathology-supported genetic testing strategy aimed at improved clinical management of MS. The study population included a total of 114 unrelated Caucasian MS patients (98 females and 16 males) and 195 unrelated Caucasian control individuals without a diagnosis of neurological disease (128 females and 67 males). A novel finding of this study was that the risk-associated FTO rs9939609 A-allele was associated with raised homocysteine levels (p=0.003) in patients diagnosed with MS, but not in controls. Furthermore, homocysteine levels correlated significantly with bo dy mass index (BMI) (p=0.046) and total cholesterol levels (p=0.048). Both homocysteine (p=0.011) and BMI (p=0.017) were significantly reduced with increasing intake of folate in the diet, while high saturated/trans fat intake correlated significantly with increased BMI (p<0.001). High physical activity correlated with reduced BMI (p<0.006) in the study population, adjusted for age, gender and disease status. Daily intake of at least five fruit and vegetable portions and the COMT rs4680 (472 G>A) AA genotype had a favourable lowering effect on MS disability as assessed by the expanded disability status scale (EDSS) (p=0.035), while smoking increased MS disability significantly (p<0.001). All SNPs studied were found to be in Hardy-Weinberg equilibrium (HWE), with no significant differences detected between patients and control individuals in genotype distribution or allele frequencies. This study has shown for the first time that the underlying disease process of MS moderates the effect of the FTO rs9939609 polymorphism on homocysteine levels , which is consistent with the role of FTO in demethylation and epigenetic changes. Identification of FTO rs9939609 reinforces the importance of adequate folate intake in the diet that can be assessed accurately with use of the Medical History and Lifestyle Questionnaire applied in this study. Finally, the finding that raised homocysteine levels and BMI are significantly influenced by lifestyle factors such as diet and physical activity in our study cohort , offers a solution to counteract the detrimental effects of genetic risk factors contributing to the development of these established vascular risk factors for MS. Combining this information with FTO rs9939609 and COMT rs4680 genotyping may in future translate into a comprehensive pathology supported genetic testing strategy aimed at improved risk management and quality of life in MS patients. / AFRIKAANSE OPSOMMING: Die etiologie van meervoudige sklerose (MS) is grootliks onbekend as gevolg van die multifaktoriale aard van siekte vatbaarheid wat bepaal word deur beide genetiese en omgewingsfaktore. Vordering is reeds gemaak in die identifisering van die genetiese component van MS, asook moontlike interaksie met die omgewing. In hierdie studie is enkel nukleotied polimorfismes (SNPs) in die FTO (rs9939609, Intron 1 T > A), MTR (rs1805087, 2756 A> G), MTRR (rs1801394, 66 A> G), MTHFR (rs1801133, 677 C > T en rs1801131, 1298 A> C) en COMT (rs4680, 472 G > A) gene, wat betrokke is in die metilering metaboliese padweg, in die konteks van MS bestudeer. Die oorhoofse doel van hierdie studie was om die onderliggende meganisme betrokke by verhoogde homosisteïen vlakke in MS pasiënte uit te lig. Die spesifieke doelwitte was 1) om die analitiese geldigheid van die hoë deurvoer riëeltyd polymerase kettingreaksie (RT-PCR) genotipering metode soos toegepas vir die 6 geselekteerde SNPs te bevestig teen direkte DNA volgorde bepaling as die goue standaard, vir 2) moontlike integrasie in 'n patologie-gesteunde genetiese toetsing (PSGT) stategie wat gemik is op verbeterde kliniese hantering van MS. Die studiepopulasie bestaan uit 'n totaal van 114 nie-verwante Kaukasiese MS pasiënte (98 vroue en 16 mans) en 195 nie-verwante Kaukasiese kontroles sonder ‘n diagnose van neurologiese siektes (128 vroue en 67 mans). 'n Nuwe bevinding van hierdie studie was dat die risiko-verwante FTO rs9939609 A- alleel geassosieer was met verhoogde homosisteïen vlakke (p = 0,003) in pasiënte gediagnoseer met MS, maar nie in kontroles nie. Homosisteïen vlakke was verder beduidend geassosieer met liggaamsmassa-indeks (BMI) (p=0,046) en totale cholesterol vlakke (p=0.048). Beide homosisteïen (p=0,011) en BMI (p=0,017) het aansienlik verminder met 'n hoër inname van folaat in die dieet, terwyl 'n hoë versadigde/trans vet en koolhidrate inname beduidend gekorreleer het met 'n verhoogde BMI (p <0.001). Hoë fisiese aktiwiteit was gekorreleer met 'n verminderde BMI (p< 0.006) in die gekombineerde groep, aangepas vir die ouderdom, geslag en MS diagnose. Daaglikse inname van ten minste vyf vrugte en groente porsies en die COMT rs4680 (472 G>A) AA genotipe het 'n gunstige uitwerking op vermindering van gestremdheid gehad, soos bepaal deur die uitgebreide gestremdheid status skaal (EDSS) (p=0,035), terwyl rook MS gestremdheid beduidend verhoog het (p <0.001). Alle SNPs bestudeer was in Hardy-Weinberg ewewig (HWE), met geen beduidende verskille waargeneem in genotipe verspreiding of alleelfrekwensies tussen pasiënte en kontroles nie. Hierdie studie het vir die eeste keer aangetoon dat ‘n diagnose van MS die effek van die FTO rs9939609 polimorfisme op homosisteïen vlakke modereer, wat ooreenstem met die rol van FTO in demetilering en epigenetiese veranderinge. Identifikasie van FTO rs9939609 versterk die belangrikheid van genoegsame folaat inname in die dieet wat akkuraat gemeet kon word deur gebruik te maak van die Mediese Geskiedenis en Leefstyl Vraelys soos toegepas in hierdie studie. Ten slotte, die bevinding dat verhoogde homosisteïen vlakke en BMI statisties betekenisvol beïnvloed word deur leefstylfaktore soos dieet en fisiese aktiwiteit in ons studie populasie, verskaf 'n oplossing om die genetiese bydrae tot hierdie gevestigde vaskulêre risikofaktore vir MS teen te werk. Kombinasie van hierdie inligting met FTO rs9939609 en COMT rs4680 genotipering kan moontlik in die toekoms benut word as deel van 'n omvattende patologie- gesteunende genetiese toetsing strategie wat daarop gemik is om die risikobestuur en kwaliteit van lewe te verbeter in MS pasiënte.

Neural tube defects (NTDs) are a world health issue, affecting approximately 1 in every 1000 live births. These congenital defects arise from the improper closure of the neural tube during development, resulting in significant, life-threatening malformations of the central nervous system. Although it has been observed that supplementing women of child-bearing age with folates greatly decreases the chances of having an NTD affected baby, unfortunately these defects still occur. It is accepted that these complex disorders arise from a combination of genetic, environmental, and dietary influences. One such dietary influence is the one-carbon metabolism metabolite, homocysteine. Homocysteine is a byproduct of methylation reactions in the cell that exists in an inverse homeostasis with folate. Homocysteine can also undergo a transformation that allows it to then react with exposed lysine or cysteine residues on proteins, in a process known as N-homocysteinylation or S-homocysteinylation respectively. High levels of homocysteine have been long correlated with many disease states, including NTDs. One potential mechanism by which homocysteine confers its negative effects is through protein N-homocysteinylation. Here, a novel and high-throughput assay for N-homocysteinylation determination is described. This assay is shown to be accurate with mass spectrometry then shown to be biologically relevant using known hyperhomocysteinemia mouse models. This assay was then applied to a cohort of neural tube closure staged mouse embryos with two different genetic mutations that have previously been shown to predispose mice to NTDs. The genotypes explored here are mutations to the LRP6 gene and the Folr1 gene, both of which have been described as folate-responsive NTD mouse models. It was seen that maternal diet and embryonic genotype had the largest influence on the developmental outcome of these embryos; however, the inverse relationship between folate and homocysteine seemed to be established at this early time point, emphasizing the importance of the balance in one-carbon metabolism. One of these genes, LRP6, was then explored in a human cohort of spina bifida cases. Four novel mutations to the LRP6 gene were found and compared to the mouse model used in the previous study. One of the mutations found in the human population was seen to mimic that of the LRP6 mouse model, therefore expanding the potential of this NTD model. / text

Homocysteine, an independent risk factor for cardiovascular disease, is methylated in the liver via the zinc metalloenzyme betaine-homocysteine methyltransferase (BHMT). Established assays for BHMT include a radiochemical assay, a colorometric assay, an HPLC assay and an in vivo microbiological assay. These techniques are either unsuitable for substrate specificity studies, or are unable to give kinetic measurements. BHMT was purified from liver and measured directly and kinetically by a novel ¹H-NMR spectroscopic assay. The disappearance of substrates and the formation of products are monitored simultaneously. Using 2 mM glycine betaine and homocysteine as substrates in 20 mM phosphate buffer (pH = 7.5) and measuring the production of N,N-dimethylglycine the CV is 6.3% (n = 6) and the detection limit is 6 nkatal. An endpoint assay for BHMT activity was also developed and had CV = 5.3%, n = 6, with a detection limit of 2 nkatal. The NMR spectroscopic assay was used to determine the substrate specificity with a library of alternative substrates. Analysis of betaine analogues with different chain length, α-substitution, substitution of the nitrogen and carboxyl moieties demonstrated that BHMT is inactive if there is any steric crowding of the nitrogen or α-carbon positions. BHMT is capable of using group VI heteroatom betaines as methyl donors, with much faster rates than glycine betaine. For glycine betaine the Km was 0.19 ± 0.03 mM with a Vmax of 17 ± 0.7 nMol min-1 mg-1. The same assay was used to detect and partially characterise a BHMT activity from hagfish liver that is similar to that of the mammalian enzyme. NMR spectroscopy was adapted for measurements of glycine betaine in urine, along with other medically significant methylamines. These were shown to be valid for clinical use and in animal studies. A novel metabolite of the sulfonium analogue of glycine betaine (methylsulfinylmethanoate) was identified in rats.

A olanzapina é um fármaco antipsicótico atípico utilizado no tratamento da esquizofrenia. Como efeitos adversos relacionados ao seu uso encontram-se obesidade, hiperlipidemia, diabetes mellitus tipo 2 e hipertensão, podendo acarretar no desenvolvimento de síndrome metabólica. O presente trabalho tem por objetivo realizar a monitorização terapêutica em pacientes na terapia com olanzapina, estabelecendo uma possível correlação entre a dose-concentração plasmática e a potenciação do risco a reações adversas, constituindo suporte clínico na eficácia do tratamento. A análise dos parâmetros antropométricos (peso, índice de massa corpóreo, circunferência do braço, circunferência do abdome, circunferência do quadril, prega cutânea triciptal, prega cutânea biciptal, prega cutânea subescapular, prega cutânea suprailíaca, resistência, porcentagem de massa gorda e taxa metabólica basal), parâmetros hemodinâmicos (pressão arterial sistólica, pressão arterial diastólica e freqüência cardíaca) e parâmetros bioquímicos (aspartato aminotranferase, alanina aminotransferase, gama glutamiltransferase, creatinina, ácido úrico, amilase, insulina, bilirrubinas, glicemia, cálcio, sódio e potássio), perfil lipídico (colesterol total, LDLcolesterol, HDL-colesterol e triglicérides), concentração de homocisteína e proteína C reativa, são relevantes na medida em que representam marcadores importantes de alterações relacionadas a desordens metabólicas ou de risco aterosclerótico. Como a obesidade e as coronariopatias podem induzir tais alterações, é importante avaliar qual o risco que o aumento de peso induzido pela olanzapina traz para o paciente esquizofrênico. A análise estatística foi realizada utilizando-se um modelo linear de efeito misto, apropriado para análise de medidas realizadas ao longo de um período pré-estabelecido. O nível de significância foi fixado em p<0,05. / The olanzapine is an atypical antipsychotic drug used to treat schizophrenia. How adverse effects related to its use, are obesity, hyperlipidemia, diabetes mellitus type II and hypertension, may result in the development of metabolic syndrome. This study aims to carry out monitoring therapy in patients in treatment with olanzapine, establishing a possible correlation between the dose-plasma concentration and potentiation of the risk to adverse reactions, providing clinical support in the effectiveness of treatment. Analysis of anthropometric parameters (weight, body mass index, arm circumference, circumference of the abdomen, the hip circumference, triceps skinfold thickness, biciptal skin fold, subscapular skin fold, skin fold suprailíaca, strength, percentage of fat mass and metabolic rate basal), hemodynamic parameters (systolic blood pressure, diastolic blood pressure and heart rate) and biochemical parameters (aspartate aminotranferase, alanine aminotransferase, gamma glutamyl transferase, creatinine, uric acid, amylase, insulin, total bilirubin, glucose, calcium, sodium and potassium ), lipid profile (total cholesterol, LDL-cholesterol, HDL-cholesterol and triglycerides), concentration of homocysteine and C-reactive protein, are relevant in so far represent important markers of changes related to metabolic disorders and atherosclerotic risk. As obesity and coronary diseases can induce such changes, which is important to evaluate the risk that the weight gain induced by olanzapine brings to the schizophrenic patient. Statistical analysis was performed using a mixed effect linear model, suitable for analysis of measurements taken over a pre-established. The significance level was set at p <0.05.

Qiao, Mu. / "June 2003." / Thesis (Ph.D.)--Chinese University of Hong Kong, 2003. / Includes bibliographical references (p. 190-209). / Electronic reproduction. Hong Kong : Chinese University of Hong Kong, [2012] System requirements: Adobe Acrobat Reader. Available via World Wide Web. / Mode of access: World Wide Web.

abstract: According to a 2016 census, eight million adults conform to a vegetarian diet within the United States, and about 50% of these adults follow a vegan diet. The census determined that plant-based diets are quickly growing in popularity particularly in young adults between the ages of 18 to 34 years. Many Americans are aware of the health benefits of a plant-based diet, however, the dietary risks associated with these diets are not well emphasized. Health concerns such as vitamin deficiencies and altered metabolism are heightened in vegetarian populations. One Particular nutrient that is commonly lacking in the vegetarian diet is vitamin B12. Vitamin B12 is found mainly in animal-derived food sources such as meat, poultry, fish, dairy, and eggs. Although some vegetarians, called lacto-ovo vegetarians, consume dairy and eggs, vegans do not consume any animal products at all. Vitamin B12 deficiency can have devastating consequences on the human body due to its role as a methylation cofactor. Metabolism, DNA replication, and cancer formation all involve methylation processes. This cross-sectional, differential study aimed to further understand the relationship between vegetarianism, vitamin B12 status, and methylation capacity in healthy adults. A group of 34 healthy adults (18 vegetarians and 16 omnivores) was recruited to analyze serum B12, homocysteine, methylmalonic acid, serum total folate, and transcobalamin II status. It was hypothesized that (1) vegetarians would have a lower vitamin B12 status, and thus, a lower methylation capacity than omnivores and that (2) low vitamin B12 status would be correlated with low methylation capacity. The data show that vegetarians did not have significantly lower vitamin B12 methylation capacity status than omnivores. Nor was vitamin B12 status correlated with methylation capacity. However, the data revealed that diet quality had a positive influence on folate status. There was also a statistical trend (p=0.08) for homocysteine reduction in participants consuming high-quality diets. The data herein suggest that methylation capacity may be impacted by the quality of diet rather than the type of diet. / Dissertation/Thesis / Masters Thesis Nutrition 2019

Hyperhomocysteinemia, a condition of elevated blood homocysteine level, is an independent risk factor for cardiovascular diseases. Folic acid can effectively reduce blood homocysteine levels. Recent studies have shown that hyperhomocysteinemia is also associated with liver disorders. However, the underlying mechanisms remain unclear. The general objective of my study was to investigate the biochemical and molecular mechanisms of homocysteine-induced liver injury and abnormal lipid metabolism. Hyperhomocysteinemia was induced in Sprague-Dawley rats by feeding a high-methionine diet for 4 weeks. An elevation of serum aminotransferases activities (indicator for liver injury) and an increase in hepatic lipid peroxidation were observed in hyperhomocysteinemic rats. Hyperhomocysteinemia-induced superoxide anion production led to oxidative stress in the liver. Reduction of oxidative stress by inhibiting superoxide anion production ameliorated hyperhomocysteinemia-induced liver injury. A significant elevation of hepatic and serum cholesterol concentrations in hyperhomocysteinemic rats was observed, exclusively due to increased expression of HMG-CoA reductase in hepatocytes. The molecular mechanisms of homocysteine-induced adverse effects were further investigated in isolated rat hepatocytes and in human hepatoma cells (HepG2). Hcy stimulated HMG-CoA reductase expression in hepatocytes via activation of transcription factors, namely, sterol regulatory element-binding protein-2 (SREBP-2), cAMP response element binding protein (CREB) and nuclear factor Y (NF-Y). Activation of these 3 transcription factors was detected in hyperhomocysteinemic rat liver and in homocysteine-treated hepatocytes. Pretreatment of hepatocytes with inhibitors for individual transcription factors effectively attenuated Hcy-induced HMG-CoA reductase mRNA expression. Supplementation of folic acid in diet significantly reduced serum homocysteine level and effectively inhibited hyperhomocysteinemia-induced superoxide anion production, resulting in amelioration of oxidative stress-mediated liver injury in hyperhomocysteinemic rats. These results reflected a protective role of folic acid in hyperhomocysteinemia-induced liver injury. In conclusion, the present study demonstrates that (1) hyperhomocysteinemia can cause oxidative stress and liver injury; (2) homocysteine stimulates cholesterol biosynthesis in hepatocytes via transcriptional regulation of HMG-CoA reductase expression; (3) supplementation of folic acid offers a hepatoprotective effect during hyperhomocysteinemia. Oxidative stress and accumulation of cholesterol in the liver contribute to liver injury associated with hyperhomocysteinemia. The role of folic acid in maintaining good health may extend beyond the cardiovascular system to encompass hyperhomocysteinemia-associated liver disorders. / October 2007

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