Candida albicans cell wall component ß-glucan has been extensively studied for its ability to induce epigenetic and functional reprogramming of innate immune cells, a process termed trained immunity. We show that a high-complexity blend of two individual ß-glucans from Saccharomyces cerevisiae possesses strong bioactivity, resulting in an enhanced trained innate immune response by human primary monocytes. The training required the Dectin-1/CR3, TLR4, and MMR receptors, as well as the Raf-1, Syk, and PI3K downstream signaling molecules. By activating multiple receptors and downstream signaling pathways, the components of this ß-glucan preparation are able to act synergistically, causing a robust secondary response upon an unrelated challenge. In in-vivo murine models of melanoma and bladder cell carcinoma, pre-treatment of mice with the ß-glucan preparation led to a significant reduction in tumor growth. These insights may aid in the development of future therapies based on ß-glucan structures that induce an effective trained immunity response.
Saccharomyces cerevisiae , beta-Glucans , Humans , Animals , Mice , Trained Immunity , beta-Glucans/pharmacology , Monocytes , Signal Transduction
Background: Histamine intolerance manifests when there is an imbalance between the production of histamine and the body's capacity to metabolise it. Within the gastrointestinal tract, diamine oxidase (DAO) plays a pivotal role in breaking down ingested histamine. Insufficient levels of DAO have been linked to various diseases affecting the respiratory, cardiovascular, nervous, muscular, and digestive systems; some of these symptoms are evidenced in fibromyalgia syndrome. This underscores the crucial role of DAO in maintaining the histamine balance and highlights its association with diverse physiological systems and health conditions. The management of fibromyalgia commonly involves the use of psychotropic medications; however, their potential interactions with DAO remain not fully elucidated. Methods: This study delved into the influence of various psychotropic medications on DAO activity through in vitro experiments. Additionally, we explored their impact on the human intestinal cell line Caco-2, examining alterations in DAO expression at both the mRNA and protein levels along with DAO activity. Results: Notably, the examined drugs-sertraline, pregabalin, paroxetine, alprazolam, and lorazepam-did not exhibit inhibitory effects on DAO activity or lead to reductions in DAO levels. In contrast, citalopram demonstrated a decrease in DAO activity in in vitro assays without influencing DAO levels and activity in human enterocytes. Conclusions: These findings imply that a collaborative approach involving psychotropic medications and DAO enzyme supplementation for individuals with fibromyalgia and a DAO deficiency could offer potential benefits for healthcare professionals in their routine clinical practice.
Long-chain dextrans are α-glucans that can be produced by lactic acid bacteria. NextDextTM, a specific long-chain dextran with a high degree of polymerisation, produced using Weissella cibaria, was recently shown to exert prebiotic potential in vitro. In this study, the ex vivo SIFR® technology, recently validated to provide predictive insights into gut microbiome modulation down to the species level, was used to investigate the effects of this long-chain dextran on the gut microbiota of six human adults that altogether covered different enterotypes. A novel community modulation score (CMS) was introduced based on the strength of quantitative 16S rRNA gene sequencing and the highly controlled ex vivo conditions. This CMS overcomes the limitations of traditional α-diversity indices and its application in the current study revealed that dextran is a potent booster of microbial diversity compared to the reference prebiotic inulin (IN). Long-chain dextran not only exerted bifidogenic effects but also consistently promoted Bacteroides spp., Parabacteroides distasonis and butyrate-producing species like Faecalibacterium prausnitzii and Anaerobutyricum hallii. Further, long-chain dextran treatment resulted in lower gas production compared to IN, suggesting that long-chain dextran could be better tolerated. The additional increase in Bacteroides for dextran compared to IN is likely related to the higher propionate:acetate ratio, attributing potential to long-chain dextran for improving metabolic health and weight management. Moreover, the stimulation of butyrate by dextran suggests its potential for improving gut barrier function and inflammation. Overall, this study provides a novel tool for assessing gut microbial diversity ex vivo and positions long-chain dextran as a substrate that has unique microbial diversity enhancing properties.
Histamine intolerance arises when there is a disparity between the production of histamine and the body's ability to break it down. In the gastrointestinal tract, the primary enzyme responsible for metabolizing ingested histamine is diamine oxidase (DAO), and a shortage of this enzyme has been associated with some diseases related to the respiratory, cardiovascular, nervous, muscular, and digestive systems, in addition to migraines. The treatment of migraines typically revolves around the utilization of both anti-migraine and anti-inflammatory drugs, but their interaction with DAO is not thoroughly understood. In this study, we examined the impact of nonsteroidal anti-inflammatory drugs (NSAIDs) and anti-migraine medications on DAO activity through in vitro experiments. We also investigated their effects on the human intestinal cell line Caco-2, assessing changes in DAO expression (both at the mRNA and protein levels) as well as DAO activity. The tested drugs, including ibuprofen, acetylsalicylic acid, paracetamol, a combination of acetylsalicylic acid with paracetamol and caffeine, zolmitriptan, and sumatriptan, did not inhibit DAO activity or reduce their levels. However, naproxen reduced DAO protein levels in human enterocyte cultures while not affecting DAO activity. These results suggest that combining anti-inflammatory and anti-migraine drugs with DAO enzyme supplementation for migraine patients with DAO deficiency could be beneficial for healthcare professionals in their daily practice.
Introducción: el carcinoma micropapilar infiltrante (CMI) es una variante histológica inusual y potencialmente agresiva caracterizada por primera vez en 1993 por Siriangkul et al. y que no formó parte de la clasificación de la Organización Mundial de la Salud (OMS) hasta 2003, como tumor mamario epitelial. Representa menos del 2% del total de carcinomas invasivos de la mama y se presupone que presenta un pronóstico desfavorable en comparación con otros carcinomas convencionales debido a su elevado tropismo vascular y linfático. Material y métodos: hasta la fecha no existe ningún estudio con un número elevado de pacientes procedentes de un único centro (> 100 casos) con un periodo de seguimiento largo (> 20 años) que compare la supervivencia del CMI con otros carcinomas convencionales no micropapilares. Se ha llevado a cabo un estudio retrospectivo, observacional con un total de 401 pacientes: 174 con CMI y 227 con otros carcinomas convencionales. Resultados: el CMI presenta mayor grado histológico, mayor afectación ganglionar y mayor riesgo de metástasis a distancia en comparación con otros carcinomas convencionales de características similares. Sin embargo, en el análisis multivariante considerando factores pronósticos como edad, tamaño tumoral, afectación ganglionar y grado histológico, no se observan diferencias estadísticamente significativas para la supervivencia global y libre de enfermedad entre los CMI diagnosticados en el mismo periodo de tiempo que los casos pareados del grupo control y otros carcinomas convencionales. Conclusión: la supervivencia global y libre de enfermedad es similar entre el CMI y otros carcinomas convencionales a igual edad, tamaño tumoral, grado histológico y afectación ganglionar. (AU)
Introduction: Invasive Micropapillary Carcinoma of the breast (IMPC) is an unusual and aggressive histological variant characterized for the first time in 1993 by Siriangkul et al. and classified by the World Health Organization in 2003 as an epithelial breast tumor. It represents less than 2% of all invasive carcinomas of the breast and is presumed to have an unfavorable prognosis compared to other conventional carcinomas due to its high vascular and lymphatic tropism. Material and methods: Until now, there is no study with a large number of patients from a single center with a long follow-up period that compares the survival of IMPC with other conventional non-micropapillary carcinomas. A retrospective, observational study has been carried out with a total of 401 patients: 174 with IMPC and 227 with other conventional carcinomas. Results: IMPC has a higher histological grade, greater lymph node involvement and a higher risk of distant metastasis compared to other conventional carcinomas. However, in the multivariate analysis considering date of diagnosis, age, tumor size, lymph node involvement and histological grade as variables, no statistically significant differences were observed for overall and disease- free survival between IMPC and other conventional carcinomas. Conclusion: Overall and disease-free survival is similar between IMPC and other conventional carcinomas considering same age, tumor size, histological grade, and lymph node involvement. (AU)
Humans , Male , Female , Adult , Middle Aged , Carcinoma, Papillary/diagnosis , Survivorship , Retrospective Studies , Longitudinal Studies , Spain
Histamine intolerance occurs when there is an imbalance between histamine production and the capacity for histamine degradation. Diamine oxidase (DAO) is the main enzyme for the catabolism of ingested histamine degradation in the gastrointestinal tract and its deficiency has been linked to allergy-like symptoms. Psychostimulant drugs are commonly used to treat Attention Deficit Hyperactivity Disorder (ADHD), but their interaction with DAO is not well characterized. In this work, we evaluated the effects of psychostimulant drugs (methylphenidate and lisdexamfetamine) on in vitro DAO activity and in the human cell line of enterocytes (Caco-2), evaluating DAO expression (mRNA and protein) and DAO activity. Methylphenidate and lisdexamfetamine did not repress the in vitro DAO activity. In addition, in Caco-2 cells, lisdexamfetamine promoted a strong upregulation of DAO mRNA levels, whereas methylphenidate tended to induce DAO activity. To sum up, methylphenidate and lisdexamfetamine treatments do not reduce DAO activity. These findings could be useful for physicians prescribing these two drugs to ADHD patients affected by DAO deficiency.
OBJECTIVE: To provide a description of laparoscopic myomectomy and the two hemostatic techniques performed over the last 11 years in a single reference center for gynecology and obstetrics and to evaluate the factors associated with favorable surgical outcomes. STUDY DESIGN: We retrospectively analyzed 625 who underwent laparoscopic myomectomy from January 2009 to December 2019. RESULTS: Of 625 patients, 437 (69.8%) were symptomatic. The most common symptoms were heavy uterine bleeding (33.2%). 188 patients (30.1%) were asymptomatic but were operated in 77 cases (12.3%) for rapid fibroid growth, 32 (5.1%) for uterine cavity distortion and, in 45 cases (8.6%), the myomectomy was indicated during a surgery for other medical reason due to its accessibility. In 173 cases (27.9%) intramyometrial adrenaline was injected and in 246 cases (39.7%) a temporary blockage of the uterus blood supply was performed. Only 35 (5.6%) patients presented complications, of which, 14 (40%) were hemorrhagic. These hemorrhagic complications were more frequent when intramyometrial adrenaline was used (5,8%) than after the temporary clipping of the uterine arteries and infundibulopelvic ligaments (0,8%; p < 0,001). In the multivariate logistic regression model, the only factor statistically associated with favorable surgical outcome was the use of temporary clipping of the uterine arteries at their origin and infundibulopelvic ligaments as hemostatic technique during the surgery. CONCLUSION: Laparoscopic myomectomy was generally safe with a high level of favorable outcomes. The temporary clipping of uterine arteries and infundibulopelvic ligaments presented fewer intraoperative bleedings compared with injecting intramyometrial adrenaline.
Gynecology , Laparoscopy , Uterine Myomectomy , Uterine Neoplasms , Female , Hemostatic Techniques , Humans , Pregnancy , Retrospective Studies , Uterine Myomectomy/adverse effects , Uterine Neoplasms/surgery
Antisense oligonucleotides (AONs) are versatile molecules that can be used to modulate gene expression by binding to RNA. The therapeutic potential of AONs appears particularly high in the central nervous system, due to excellent distribution and uptake in brain cells, as well as good tolerability in clinical trials thus far. Nonetheless, immune stimulation in response to AON treatment in the brain remains a concern. For this reason we performed RNA sequencing analysis of brain tissue from mice treated intracerebroventricularly with phosphorothioate, 2'-O-methyl modified AONs. A significant upregulation of immune system associated genes was observed in brains of AON treated mice, with the striatum showing largest transcriptional changes. Strongest upregulation was seen for the antiviral enzyme 2'-5'-oligoadenylate synthase-like protein 2 (Oasl2) and Bone marrow stromal antigen 2 (Bst2). Histological analysis confirmed activation of microglia and astrocytes in striatum. The upregulation of immune system associated genes was detectable for at least 2 months after the last AON administration, consistent with a continuous immune response to the AON.
Brain/drug effects , Immunity, Innate/drug effects , Infusions, Intraventricular , Oligonucleotides, Antisense/administration & dosage , Phosphorothioate Oligonucleotides/administration & dosage , 2',5'-Oligoadenylate Synthetase/genetics , 2',5'-Oligoadenylate Synthetase/metabolism , Animals , Antigens, CD/genetics , Antigens, CD/metabolism , Astrocytes/drug effects , Brain/pathology , Membrane Glycoproteins/genetics , Membrane Glycoproteins/metabolism , Mice , Mice, Inbred C57BL , Microglia/drug effects , RNA/genetics , RNA/metabolism , Sequence Analysis, RNA
Au- and iron-based magnetic nanoparticles (NPs) are promising NPs for biomedical applications due to their unique properties. The combination of a gold coating over a magnetic core puts together the benefits from adding the magnetic properties to the robust chemistry provided by the thiol functionalization of gold. Here, the use of Au-coated magnetic NPs for molecular detection of a single methylation in DNA aptamer is described. Binding of α-thrombin to two aptamers conjugated to these NPs causes aggregation, a phenomenon that can be observed by UV, DLS and MRI. These techniques discriminate a single methylation in one of the aptamers, preventing aggregation due to the inability of α-thrombin to recognize it. A parallel study with gold and ferromagnetic NPs is detailed, concluding that the Au coating of FexOy NP does not affect their performance and that they are suitable as complex biosensors. These results prove the high detection potency of Au-coated SPIONs for biomedical applications especially for DNA repair detection.
Aptamers, Nucleotide/chemistry , Biosensing Techniques , Gold , Metal Nanoparticles , Aptamers, Nucleotide/metabolism , DNA Repair , Ferric Compounds/chemistry , Gold/chemistry , Humans , Magnetic Resonance Imaging , Metal Nanoparticles/chemistry , Metal Nanoparticles/ultrastructure , Protein Binding , Thrombin/metabolism
O(6)-alkylguanine-DNA-alkyltransferase (hAGT) activity provides resistance to cancer chemotherapeutic agents and its inhibition enhances chemotherapy. We herein present the development of a novel fluorescence assay for the detection of hAGT activity. We designed a dsDNA sequence containing a fluorophore-quencher pair, where the fluorophore was attached to an O(6)-benzylguanine. This precursor was synthesized using the Mitsunobu reaction to introduce the benzyl group. The alkyl-fluorophore group is transferred to the active site during the dealkylation, producing an increase in fluorescence which is correlated to hAGT activity. This assay can be used for the evaluation of potential inhibitors of hAGT in a straightforward manner.
Enzyme Assays/methods , Fluorescent Dyes/chemical synthesis , O(6)-Methylguanine-DNA Methyltransferase/analysis , Oligonucleotides/chemical synthesis , Base Pair Mismatch , Fluoresceins/chemistry , Fluorescent Dyes/chemistry , Humans , Oligonucleotides/chemistry
Gamma-aminobutyric acid (GABA) is the main inhibitory neurotransmitter in the central nervous system, and diseases that associate a deficiency in GABA might benefit from GABAergic drugs. Cerebellar Purkinje cells employ GABA as a neurotransmitter. Cortical cerebellar atrophy (CCA) shows Purkinje cell loss, and ataxia caused by it was alleviated by gabapentin and pregabalin. Thus, CCA is proposed as a model of selective deficiency in GABA in the cerebellum, which benefits clinically from administration of GABAergic drugs, in a manner similar in which levodopa improves motor manifestations in Parkinson's disease. Other ataxias also benefited clinically from GABAergic drugs, as adult-onset GM2 gangliosidosis, olivopontocerebellar atrophy, cerebellar ataxia with hypogonadism, spinocerebellar ataxias 1, 2 and 6, and adult-onset ataxia-telangiectasia. Complex neurochemical diseases, as multiple-system atrophy, had ataxia worsened by GABAergic drugs. Various disorders with a deficiency in GABA content had their manifestations relieved by admistration of GABAergic drugs, as one patient with progressive encephalomyelitis with rigidity, whose muscular spasms were suppressed by a combination of gabapentin and tiagabine, and another with diaphragmatic myoclonus, who required gabapentin and tiagabine for symptomatic control. On the contrary, GABAergic drugs were not effective in cervical dystonia, amyotrophic lateral sclerosis, Parkinson's disease and progressive supranuclear palsy, presumably because a deficiency in GABA is not an essential neurochemical abnormality in these diseases. Research aimed at identifying effective therapies to treat cerebellar ataxias and other motor disorders of the central nervous system is warranted. Meanwhile, therapeutic tests with GABAergic drugs might yield clinical improvement in these diseases.
Central Nervous System Diseases/drug therapy , Motor Disorders/drug therapy , gamma-Aminobutyric Acid/metabolism , Adult , Animals , Central Nervous System Diseases/physiopathology , Cerebellar Ataxia/drug therapy , Cerebellar Ataxia/physiopathology , GABA Agents/pharmacology , GABA Agents/therapeutic use , Humans , Motor Disorders/physiopathology , Neurotransmitter Agents/metabolism , Purkinje Cells/metabolism
DNA's remarkable molecular recognition properties, flexibility, and structural features make it one of the most promising scaffolds to design a variety of nanostructures. During recent decades, two major methods have been developed for the construction of DNA nanomaterials in a programmable way; both generate nanostructures in one, two, and three dimensions. The tile-based assembly process is a useful tool to construct large and simple structures; the DNA origami method is suitable for the production of smaller, more sophisticated and well-defined structures. Proteins, nanoparticles and other functional elements have been specifically positioned into designed patterns on these structures. They can also act as templates to study chemical reactions, help in the structural determination of proteins, and be used as platform for genomic and drug delivery applications. In this review we examine recent progresses towards the potential use of DNA nanostructures in molecular and cellular biology.
DNA/chemistry , Nanostructures/chemistry , Animals , Biosensing Techniques/instrumentation , Biosensing Techniques/methods , DNA/metabolism , Humans , Models, Molecular , Nanostructures/ultrastructure , Nanotechnology/instrumentation , Nanotechnology/methods , Nucleic Acid Conformation
The folding of DNA molecules by DNA origami is used in a nanosensor to analyze enzymatic DNA repair activity of hAGT. The method uses conformational changes that condition α-thrombin interaction with DNA aptamers, and illustrates the use of DNA origami as a proteinrecognition biosensor.
Angiotensinogen/metabolism , Aptamers, Nucleotide , Biosensing Techniques , DNA Repair , Nanostructures , Nanotechnology , Thrombin/metabolism , Aptamers, Nucleotide/chemistry , Humans , Microscopy, Atomic Force , Nanostructures/chemistry
The endonucleolytic activity of human apurinic/apyrimidinic endonuclease (AP endo, Ape1) is a major factor in maintaining the integrity of the genome. Conversely, as an undesired effect, Ape1 overexpression has been linked to resistance to radio- and chemotherapeutic treatments in several human tumors. Inhibition of Ape1 using siRNA or the expression of a dominant negative form of the protein has been shown to sensitize cells to DNA-damaging agents, including various chemotherapeutic agents. Therefore, inhibition of the enzymatic activity of Ape1 might result in a potent antitumor therapy. A number of small molecules have been described as Ape1 inhibitors; however, those compounds are in the early stages of development. Herein we report the identification of new compounds as potential Ape1 inhibitors through a docking-based virtual screening technique. Some of the compounds identified have in vitro activities in the low-to-medium micromolar range. Interaction of these compounds with the Ape1 protein was observed by mass spectrometry. These molecules also potentiate the cytotoxicity of the chemotherapeutic agent methyl methanesulfonate in fibrosarcoma cells. This study demonstrates the power of docking and virtual screening techniques as initial steps in the design of new drugs, and opens the door to the development of a new generation of Ape1 inhibitors.
DNA-(Apurinic or Apyrimidinic Site) Lyase/antagonists & inhibitors , Drug Design , Enzyme Inhibitors/chemistry , Enzyme Inhibitors/pharmacology , Catalytic Domain , Cell Line, Tumor , Cell Survival/drug effects , DNA/metabolism , DNA-(Apurinic or Apyrimidinic Site) Lyase/chemistry , DNA-(Apurinic or Apyrimidinic Site) Lyase/metabolism , Humans , Intercalating Agents/chemistry , Intercalating Agents/pharmacology , Molecular Docking Simulation , Neoplasms/drug therapy
The thrombin binding aptamer (TBA) is a well characterized chair-like, antiparallel quadruplex structure that binds specifically to thrombin at nanomolar concentrations and therefore it has interesting anticoagulant properties. In this article we review the research involved in the development of new TBA derivatives with improved anticoagulant properties as well as the use of the TBA as a model compound for the study of quadruplex structures. Specifically, we describe the impact of modified nucleosides and non-natural backbones in the guanine tetrads or in the loops and the introduction of pendant groups at the 3' or 5'-ends. The modified oligonucleotides are shown to be excellent tools for the understanding of the molecular structure of the TBA and its folding properties. Finally, we review the use of the TBA-Thrombin recognition system for the development of analytical tools based on the TBA folding.
Anticoagulants/chemistry , Aptamers, Nucleotide/chemistry , Anticoagulants/pharmacology , Aptamers, Nucleotide/pharmacology , Biosensing Techniques , Clinical Trials as Topic , G-Quadruplexes , Humans , Magnetic Resonance Spectroscopy , Nucleic Acid Conformation , Structure-Activity Relationship , Thrombin/chemistry
The aim of the study was to enhance our understanding of the pathogenesis of the ataxia of Charlevoix-Saguenay, based on the findings presented herein. Five patients with a molecular diagnosis of this disease underwent clinical, radiological, ophthalmologic and electrophysiological examinations. Five novel mutations, which included nonsense and missense variants, were identified, with these resulting in milder phenotypes. In addition to the usual manifestations, a straight dorsal spine was found in every case, and imaging techniques showed loss of the dorsal kyphosis. Cranial MRI demonstrated hypointense linear striations at the pons. Tensor diffusion MRI sequences revealed that these striations corresponded with hyperplastic pontocerebellar fibres, and tractographic sequences showed interrupted pyramidal tracts at the pons. Ocular coherence tomography demonstrated abnormal thickness of the nerve fibre layer. Electrophysiological studies showed nerve conduction abnormalities compatible with a dysmyelinating neuropathy, with signs of chronic denervation in distal muscles. The authors suggest that the hyperplastic pontocerebellar fibres compress the pyramidal tracts at the pons, and that the amount of retinal fibres traversing the optic discs is enlarged. These facts point to the contribution of an abnormal developmental mechanism in the ataxia of Charlevoix-Saguenay. Accordingly, spasticity would be mediated by compression of the pyramidal tracts, neuromuscular symptoms by secondary axonal degeneration superimposed on the peripheral myelinopathy, while the cause of the progressive ataxia remains speculative. The distinctive aspect of the dorsal spine could be of help in the clinical diagnosis.
Brain/pathology , Muscle Spasticity/pathology , Muscle Spasticity/physiopathology , Spinocerebellar Ataxias/congenital , Adult , Diffusion Magnetic Resonance Imaging , Disease Progression , Female , Heat-Shock Proteins/genetics , Humans , Male , Middle Aged , Muscle Spasticity/diagnostic imaging , Muscle Spasticity/genetics , Mutation/genetics , Neural Conduction/physiology , Radiography , Spinocerebellar Ataxias/diagnostic imaging , Spinocerebellar Ataxias/genetics , Spinocerebellar Ataxias/pathology , Spinocerebellar Ataxias/physiopathology , Tomography, Optical Coherence
The autosomal recessive spastic ataxia of Charlevoix-Saguenay (ARSACS) is considered a neurodegenerative disease caused by mutations in the SACS gene, located on chromosome 13q12.12. It is a syndrome that comprises skeletal, retinal and neurological manifestations, among which feature spasticity, cerebellar ataxia and peripheral neuropathy. Five patients with a molecular diagnosis of ARSACS underwent clinical, radiological, and ophthalmologic examinations. Every one of the identified causal mutations was novel. Spastic ataxia, peripheral neuropathy, pes cavus, and hammertoes were found in every case. T2 and T2-fluid attenuation inversion recovery-weighted MRI sequences demonstrated cerebellar atrophy and a hypointense linear striation at the pons. Tensor diffusion sequences revealed that the hypointense striation corresponded with hyperplasia of the pontocerebellar fibres, which gave place to abnormally thick middle cerebellar peduncles. Stereophotographs of the optic discs showed an increased number of retinal fibres, and ocular coherence tomography, increased thickness of the retinal nerve fibre layer. The authors suggest that the hyperplasic pontocerebellar fibres compress the pyramidal tracts at the pons since a very early stage of central nervous system development, causing spasticity, and may also cause cerebellar atrophy by means of glutamate-induced excitotoxicity. The abnormal amount of retinal fibres traversing the optic discs could have caused the detected mild peripheral visual field defects. Taken together, these facts point to a developmental cause in ARSACS, as it does not exhibit the tissue atrophy characteristic of degenerative diseases. Clinical deterioration in ARSACS seems to be mediated by phenomena (compression of the pyramidal tracts and cerebellar glutamate-mediated excitotoxicity) derived from the developmental anomalies referred to, while the neuromuscular symptoms are caused by a peripheral neuropathy with pathologic features suggestive of a similar origin. These observations should be taken into account when research about the origin of ARSACS is undertaken.
Cerebellum/physiopathology , Heat-Shock Proteins/genetics , Muscle Spasticity/genetics , Muscle Spasticity/pathology , Optic Disk/physiopathology , Spinocerebellar Ataxias/congenital , Adult , Cerebellum/diagnostic imaging , Cerebellum/growth & development , Diffusion Tensor Imaging , Humans , Magnetic Resonance Imaging , Male , Middle Aged , Mutation/genetics , Optic Disk/growth & development , Radiography , Sequence Analysis, DNA , Spinocerebellar Ataxias/genetics , Spinocerebellar Ataxias/pathology
Human O(6)-alkylguanine-DNA alkyltransferase (hAGT) is a DNA repair protein that reverses the effects of alkylating agents by removing DNA adducts from the O(6) position of guanine. Here, we developed a real-time fluorescence hAGT activity assay that is based on the detection of conformational changes of the thrombin-binding aptamer (TBA). The quadruplex structure of TBA is disrupted when a central guanine is replaced by an O(6)-methyl-guanine. The sequence also contains a fluorophore (fluorescein) and a quencher (dabsyl) attached to the opposite ends. In the unfolded structure, the fluorophore and the quencher are separated. When hAGT removes the methyl group from the central guanine of TBA, it folds back immediately into its quadruplex structure. Consequently, the fluorophore and the quencher come into close proximity, thereby resulting in decreased fluorescence intensity. Here, we developed a new method to quantify the hAGT without using radioactivity. This new fluorescence resonance energy transfer assay has been designed to detect the conformational change of TBA that is induced by the removal of the O(6)-methyl group.
