7 research outputs found
Probes of tocopherol biochemistry: fluorophores, imaging agents, and fake antioxidants
The body has many defence systems against reactive radical species, but none are as crucial in
the protection of lipid membranes as vitamin E. As a result of a selection process mediated by
the α-tocopherol transfer protein (α-TTP), α-tocopherol is the only form of vitamin E retained
in the body. This chaperon protein has been well studied because of its role in vitamin E
transport. Furthermore, malfunctions of α-TTP cause vitamin E deficiency leading to ataxia and
other neurodegenerative disease. Protection of neuronal tissue is critical and is reflected in the
high retention of α-tocopherol in the central nervous system. Neuronal tissues receive α
tocopherol from astrocytes, cells that are linked to hepatic tissue and able to express α-TTP,
however the exact path of delivery between these cells is still unclear.
A technique called fluorescent microscopy allows the tracking of fluorescent molecules in cells
to find their location and interactions with other parts of the cell. The focus of this study is the
synthesis of a fluorescent tocopherol analogue with a long absorption wavelength, high photostability, and that binds selectively to
 α-TTP with high affinity.
Most health benefits associated with vitamin E consumption are based on its capability to
inhibit lipid peroxidation in cell membranes by scavenging reactive oxygen species (ROS).
Oxidative damage in membranes puts cells in a “stressful” state, activating signalling events
that trigger apoptosis. Vitamin E down-regulates apoptotic functions like inflammation,
macrophage activation and cell arrest in a stressed state, returning the cell back to normal
functioning. At the same time, vitamin E has a preventive effect for atherosclerosis,
Alzheimer’s and cancer.
With the deeper understanding of cell signalling processes associated with vitamin E the
question arose whether protein interactions or the ROS scavenging is responsible for cell
survival. To test this hypothesis, a non-antioxidant but α-TTP binding tocopherol analogue was
synthesized and administered into oxidatively stressed, α-TTP deficient cells. If the cells were
unable to restore homeostasis and stop apoptosis with the new molecule, this would suggest that
the antioxidant function of α-tocopherol is the reason for survival.
Cancer is regarded as one of the most detrimental diseases with a high mortality rate. One key
aspect in medical research is the increased drug specificity towards targeting cancer.
Chemotherapy applies cytotoxic compounds, which weaken the immune system because both
malignant and healthy cells are destroyed. The specificity of the anti-cancer drugs are enhanced
when encapsulated into liposomes that bear target-directing molecules such as antibodies which
recognize cancer cell specific antigens on the cell membrane. The question remains if the
encapsulated drug reaches the cancer or not.
Magnetic resonance imaging (MRI) and computed tomography (CT) are used to find malignant
tissue in the body. CT imaging uses highly charged X-ray particles to scan the patient, possibly
having damaging cytotoxic effects. Obtaining MRI results require the use of contrast agents to
enhance the quality of images. These agents are based on transition metals, which potentially
have chronic toxicity when retained in the body. Alternatively short-lived radiotracers that emit
a Îł-photon upon positron decay are used through a process called positron emission tomography
(PET). Rapid decay times make the use of PET a less toxic alternative, however the decay
products might be toxic to the cell.
For this reason a vitamin E based PET agent was created, which produces naturally safe decay
products based on known metabolites of vitamin E, useful to track liposomal delivery of
chemotherapeutic agents. This work describes the non-radioactive synthetic procedures towards
a variety of vitamin E PET analogues. The cytotoxicity of the most promising vitamin E PET
tracer was evaluated along with its synthetic byproducts
Vitamin E-inspired multi-scale imaging agent
© 2018 Elsevier Ltd The production and use of multi-modal imaging agents is on the rise. The vast majority of these imaging agents are limited to a single length scale for the agent (e.g. tissues only), which is typically at the organ or tissue scale. This work explores the synthesis of such an imaging agent and discusses the applications of our vitamin E-inspired multi-modal and multi-length scale imaging agents TB-Toc ((S,E)-5,5-difluoro-7-(2-(5-((6-hydroxy-2,5,7,8-tetramethylchroman-2-yl) methyl) thiophen-2-yl) vinyl)-9-methyl-5H-dipyrrolo-[1,2-c:2’,1’-f][1,3,2]diazaborinin-4-ium-5-uide). We investigate the toxicity of TB-Toc along with the starting materials and lipid based delivery vehicle in mouse myoblasts and fibroblasts. Further we investigate the uptake of TB-Toc delivered to cultured cells in both solvent and liposomes. TB-Toc has low toxicity, and no change in cell viability was observed up to concentrations of 10 mM. TB-Toc shows time-dependent cellular uptake that is complete in about 30 min. This work is the first step in demonstrating our vitamin E derivatives are viable multi-modal and length scale diagnostic tools
A High-Throughput Assay for In Vitro Determination of Release Factor-Dependent Peptide Release from a Pretermination Complex by Fluorescence Anisotropy—Application to Nonsense Suppressor Screening and Mechanistic Studies
Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren’s mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren’s binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren’s low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity
A High-Throughput Assay for In Vitro Determination of Release Factor-Dependent Peptide Release from a Pretermination Complex by Fluorescence Anisotropy—Application to Nonsense Suppressor Screening and Mechanistic Studies
Premature termination codons (PTCs) account for ~12% of all human disease mutations. Translation readthrough-inducing drugs (TRIDs) are prominent among the several therapeutic approaches being used to overcome PTCs. Ataluren is the only TRID that has been approved for treating patients suffering from a PTC disease, Duchenne muscular dystrophy, but it gives variable readthrough results in cells isolated from patients suffering from other PTC diseases. We recently elucidated ataluren’s mechanism of action as a competitive inhibitor of release factor complex (RFC) catalysis of premature termination and identified ataluren’s binding sites on the ribosome responsible for such an inhibition. These results suggest the possibility of discovering new TRIDs, which would retain ataluren’s low toxicity while displaying greater potency and generality in stimulating readthrough via the inhibition of termination. Here we present a detailed description of a new in vitro plate reader assay that we are using both to screen small compound libraries for the inhibition of RFC-dependent peptide release and to better understand the influence of termination codon identity and sequence context on RFC activity
The tocopherol transfer protein mediates vitamin E trafficking between cerebellar astrocytes and neurons.
Alpha-tocopherol (vitamin E) is an essential nutrient that functions as a major lipid-soluble antioxidant in humans. The alpha-tocopherol transfer protein (TTP) binds α-tocopherol with high affinity and selectivity and regulates whole-body distribution of the vitamin. Heritable mutations in the TTPA gene result in familial vitamin E deficiency, elevated indices of oxidative stress, and progressive neurodegeneration that manifest primarily in spinocerebellar ataxia. Although the essential role of vitamin E in neurological health has been recognized for over 50 years, the mechanisms by which this essential nutrient is transported in the central nervous system are poorly understood. Here we found that, in the murine cerebellum, TTP is selectively expressed in glial fibrillary acidic protein-positive astrocytes, where it facilitates efflux of vitamin E to neighboring neurons. We also show that induction of oxidative stress enhances the transcription of the TtpA gene in cultured cerebellar astrocytes. Furthermore, secretion of vitamin E from astrocytes is mediated by an ABC-type transporter, and uptake of the vitamin into neurons involves the low-density lipoprotein receptor-related protein 1. Taken together, our data indicate that TTP-expressing astrocytes control the delivery of vitamin E from astrocytes to neurons, and that this process is homeostatically responsive to oxidative stress. These are the first observations that address the detailed molecular mechanisms of vitamin E transport in the central nervous system, and these results have important implications for understanding the molecular underpinnings of oxidative stress-related neurodegenerative diseases
Vitamin E Circular Dichroism Studies: Insights into Conformational Changes Induced by the Solvent’s Polarity
We used circular dichroism (CD) to study differences in CD spectra between α-, δ-, and methylated-α-tocopherol in solvents with different polarities. CD spectra of the different tocopherol structures differ from each other in intensity and peak locations, which can be attributed to chromanol substitution and the ability to form hydrogen bonds. In addition, each structure was examined in different polarity solvents using the Reichardt index—a measure of the solvent’s ionizing ability, and a direct measurement of solvent–solute interactions. Differences across solvents indicate that hydrogen bonding is a key contributor to CD spectra at 200 nm. These results are a first step in examining the hydrogen bonding abilities of vitamin E in a lipid bilayer