Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins call... more Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins called caveolins. The presence of caveolae and caveolins in mixed cultures of human neurons and glia has not been investigated. Here, we sought to determine the presence of caveolae and caveolins in human NTera-2 (NT2/D1) cells, differentiated with retinoic acid into neuron-like (NT2/N) and astrocyte-like (NT2/A) cells. We found that while caveolin-3 mRNA levels remained relatively constant, caveolin-1 and -2 levels were upregulated in NT2/A and downregulated in NT2/N. No caveolin-1 immunoreactivity was detected in NT2/N. Electron microscopy revealed numerous flask-shaped invaginations (~86–102 nm in diameter) in the plasma membrane of NT2/A and NT2/N cells, while only few were detected in NT2/D1 cells. Immunoelectron microscopy localized caveolin-1 gold particles in the flask-shaped structures on plasmalemma and cytoplasmic vesicles of NT2/A cells. Furthermore, NT2/A endocytosed Alexa 488 c...
The world continues a desperate search for therapies that could bring hope and relief to millions... more The world continues a desperate search for therapies that could bring hope and relief to millions suffering from progressive neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s (PD). With oxidative stress thought to be a core stressor, interests have long been focused on applying redox therapies including coenzyme-Q10. Therapeutic use has failed to show efficacy in human clinical trials due to poor bioavailability of this lipophilic compound. A nanomicellar, water-dispersible formulation of coenzyme-Q10, Ubisol-Q10, has been developed by combining coenzyme-Q10 with an amphiphilic, self-emulsifying molecule of polyoxyethanyl α-tocopheryl sebacate (derivatized vitamin E). This discovery made possible, for the first time, a proper assessment of the true therapeutic value of coenzyme-Q10. Micromolar concentrations of Ubisol-Q10 show unprecedented neuroprotection against neurotoxin exposure in in vitro and in vivo models of neurodegeneration and was extremely effective wh...
The coronavirus disease 2019 (COVID-19), a serious respiratory illness caused by the severe acute... more The coronavirus disease 2019 (COVID-19), a serious respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2), has emerged as a global pandemic. Canada reported its first case of COVID-19 on the 25th January 2020. By March 2020, the virus had spread within Canadian communities reaching the most frail and vulnerable elderly population in long-term care facilities. The majority of cases were reported in the provinces of Quebec, Ontario, Alberta, and British Columbia, and the highest mortality was seen among individuals aged 65 years or older. Canada has the highest prevalence and incidence rates of several chronic inflammatory diseases, such as multiple sclerosis, inflammatory bowel disease, and Parkinson’s disease. Many elderly Canadians also live with comorbid medical illnesses, such as hypertension, diabetes, cardiovascular disease, and chronic lung disease, and are more likely to suffer from severe COVID-19 with a poor prognosis. It is becoming ...
Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essen... more Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood-brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. A constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB. Several other BBB-expressed receptors have been shown to mediate transcytosis of ligands including insulin receptor (INSR) and insulin-like growth factor-1 receptor (IGF1R), lipid transporters LRP1, LDLR, LRP8 and TMEM30A, solute carrier family transporter LAT1 (subunit CD98hc) and leptin receptor (LEPR). In this study, we analyzed expression patterns of genes encoding RMT receptors in isolated brain microvessels, the brain and peripheral organs of the mouse and the human using RNAseq approach. IGF1R, INSR and LRP8 were highly enriched in the mouse brain microvessels compared to periph...
The immature embryos of Gossypium arboreum cv. RG8 were co-bombarded with two plasmids H1Z and pB... more The immature embryos of Gossypium arboreum cv. RG8 were co-bombarded with two plasmids H1Z and pBI121 using Biolistic® PDS-1000/He Particle Delivery System. The two plasmids were coated onto tungsten particles in the ratio 1:1 at a concentration of 100ng/μl which was 10 times less than recommended in the standard protocol. A total of 1827 embryos were bombarded in 12 different experiments. The representative embryos from each bombardment experiment were analysed 48 hr post bombardment through beta glucuronidase assay called GUS assay and PCR. The histochemical results showed indigo colouration in the representative embryos from 11 experiments (91.6%). The gene specific PCR analysis on genomic DNA isolated 48 hr post bombardment from 12 representative bombarded embryos revealed 1764bp amplicon corresponding to β-1, 3-glucanase ORF in nine (75%) bombarded embryos. The results confirmed successful co-bombardment of two transgenes despite using ten times less concentration of plasmid DNA.
A major emphasis in breeding for iron toxicity tolerance in rice is to identify differences that ... more A major emphasis in breeding for iron toxicity tolerance in rice is to identify differences that are associated with resistance and harness them for genetic improvement. In this study, thirty accessions, including IRRI gene bank accessions, two varieties from Brazil, 8 cultivars from West Africa and 10 cultivars from Uganda were analyzed for sensitivity to iron toxicity, and genetic diversity using morphological and SSR markers. Two genotypes, IR61612-313-16-2-2-1 and Suakoko 8 showed significantly high resistance with an average score of ≤ 3.5 on 1-9 scale. The SRR markers were highly informative and showed mean polymorphism information content (pic) of 0.68. The PIC values revealed that RM10793, RM3412, RM333, RM562, RM13628, RM310, RM5749, and RM154 could be the best markers for genetic diversity estimation of these rice cultivars. Diversity at the gene level showed an average of 4.61 alleles ranging from 2 to 12 per locus. Mean gene diversity (H) value for all SSR loci for the 30 genotypes evaluated was 0.69 but was decreased to 0.53 when analysis was performed on Ugandan accessions. The low genetic diversity found among the Ugandan accessions is the evidence of a narrow genetic base, and such a scenario has a potential vulnerability for resistance break down. A low correlation was detected between the observed molecular and morphological datasets. This means that a combination of morphological traits and SSR analysis would be required when assessing genetic variation under iron toxic conditions, and could be a practical strategy for breeders when planning crosses. A distinction between the resistant and susceptible accessions in both phenotyping and SSR datasets suggests the presence of unique alleles that could be harnessed for improvement of rice against iron toxicity.
Because nitric oxide related species have been found in the inflamed joints of patients with arth... more Because nitric oxide related species have been found in the inflamed joints of patients with arthritis, we investigated whether protein nitrotyrosine (a marker of tissue exposure to peroxynitrite) is present in their synovial tissues. Protein nitrotyrosine was detected immunohistochemically and by Western blot analysis. Synovial tissues removed surgically from 12 patients with rheumatoid arthritis (RA) (mean age 63.7 yrs) and 20 with osteoarthritis (OA) (mean age 66.6 yrs) were studied. Nitrated proteins were detected immunohistochemically in all of 18 tissues examined. Diffuse staining of the stroma was seen in all patients, with more extensive staining in RA than OA (p = 0.008). Intense staining was detected in some lymphocytes, but not in others, even within a single lymphoid aggregate. Neutrophils did not stain for nitrotyrosine. Vascular endothelial cells stained for nitrotyrosine but adjoining smooth muscle cells did not. Both cytoplasmic and nuclear staining was seen in macro...
The role of an A/T-rich positive regulatory region (P268, −444 to −177 from the translation start... more The role of an A/T-rich positive regulatory region (P268, −444 to −177 from the translation start site) of the pea plastocyanin gene (PetE) promoter has been investigated in transgenic plants containing chimeric promoters fused to the β-glucuronidase (GUS) reporter gene. This region enhanced GUS expression in leaves of transgenic tobacco plants when fused in either orientation to a minimal pea PetE promoter (−176 to +4) and in roots when fused in either orientation upstream or downstream of a minimal cauliflower mosaic virus 35S promoter (−90 to +5). The region was also able to enhance GUS expression in microtubers of transgenic potato plants when placed in either orientation upstream of a minimal class I patatin promoter (−332 to +14). Dissection of P268 revealed that cis elements responsible for enhancing GUS expression from the minimal PetE promoter were distributed throughout P268.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1999
A new mouse model (Mutatect) that permits detection of mutations at the hprt (hypoxanthine phosph... more A new mouse model (Mutatect) that permits detection of mutations at the hprt (hypoxanthine phosphoribosyltransferase) locus is described. It is highly sensitive to detection of mutants induced by clastogenic agents such as ionizing radiation. MN-11 cells are grown as a subcutaneous tumour in C57BL/6 mice for a period of 2 weeks, during which time they can be exposed to mutagenic treatments. Cells taken from the animal are cultured ex vivo and 6-thioguanine (6-TG)-resistant mutant clones can be readily identified and scored. This model system may have special utility for detecting multi-locus deletion events (chromosomal mutations) induced by high LET forms of radiation that might be encountered in space.
The potential pathogenicity of two homoplasmic mtDNA point mutations, 9035TϾC and 4452TϾC, found ... more The potential pathogenicity of two homoplasmic mtDNA point mutations, 9035TϾC and 4452TϾC, found in a family afflicted with maternally transmitted cognitive developmental delay, learning disability, and progressive ataxia was evaluated using transmitochondrial cybrids. We confirmed that the 4452TϾC transition in tRNA Met represented a polymorphism; however, 9035TϾC conversion in the ATP6 gene was responsible for a defective F 0-ATPase. Accordingly, mutant cybrids had a reduced oligomycin-sensitive ATP hydrolyzing activity. They had less than half of the steady-state content of ATP and nearly an 8-fold higher basal level of reactive oxygen species (ROS). Mutant cybrids were unable to cope with additional insults, i.e., glucose deprivation or tertiary-butyl hydroperoxide, and they succumbed to either apoptotic or necrotic cell death. Both of these outcomes were prevented by the antioxidants CoQ 10 and vitamin E, suggesting that the abnormally high levels of ROS were the triggers of cell death. In conclusion, the principal metabolic defects, i.e., energy deficiency and ROS burden, resulted from the 9035TϾC mutation and could be responsible for the development of clinical symptoms in this family. Furthermore, antioxidant therapy might prove helpful in the management of this disease.
Parkinson's disease arises from a combination of environmental and genetic risk factors. At p... more Parkinson's disease arises from a combination of environmental and genetic risk factors. At present neither the curative nor preventative therapies are available; hence, there is an urgent need to develop reliable animal models to facilitate their development. Water soluble nanomiceller formulation of CoQ10 (Ubisol-Q10) has shown neuroprotection against neurotoxin on human neuronal cells. We have combined the genetic deficiency of DJ-1/PARK7 mice with MPTP exposure and develop a genetic susceptibility model of PD and evaluated the neuroprotective efficacy of (Ubisol-Q10). Transgenic mice with DJ-1 deficiency (DJ-1/PARK7) were given either water or Ubisol-Q10 prophylactically at a dose of 6 mg/kg/day added directly to a drinking water for one month followed challenged with MPTP injections while keeping the same drinking water regiments. Four weeks after the last injection we evaluated neuroprotective efficacy of Ubisol-Q10 in DJ-1/MPTP model of PD using histochemical and behavior...
Expression of the human respiratory syncytial virus (RSV) fusion protein (F) gene under the contr... more Expression of the human respiratory syncytial virus (RSV) fusion protein (F) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter was analyzed by enzyme-linked immunosorbent assay (ELISA) in polyethylene glycol-transfected apple leaf protoplasts. In particular, we examined whether RSV-F gene expression could be enhanced by addition of a viral leader and a plant enhancer to the chimeric gene construct. Insertion of the 5′-untranslated leader from alfalfa mosaic virus (AMV) RNA 4 between the CaMV 35S promoter and the RSV-F gene increased viral expression by 5.5-fold compared to the construct without the leader. The addition of a transcriptional enhancer from the pea plastocyanin gene (PetE) upstream of the CaMV 35S promoter to a construct containing the AMV leader further increased RSV-F gene expression by 1.4-fold. Immunoblot assays showed that the RSV-F expressed in transfected apple protoplasts reacted with RSV-F monoclonal antibodies and was of the expected molecular mass of 68 kDa. These results demonstrated that the RSV-F recombinant protein was expressed in an antigenic form in plant cells. Furthermore, protein expression was enhanced by modifying the transfection vector using both a leader and an enhancer linked to a promoter.
Although the support for the use of antioxidants, such as coenzyme Q 10 (CoQ 10), to treat Parkin... more Although the support for the use of antioxidants, such as coenzyme Q 10 (CoQ 10), to treat Parkinson's disease (PD) comes from the extensive scientific evidence, the results of conducted thus far clinical trials are inconclusive. It is assumed that the efficacy of CoQ 10 is hindered by insolubility, poor bioavailability, and lack of brain penetration. We have developed a nanomicellar formulation of CoQ 10 (Ubisol-Q 10) with improved properties, including the brain penetration, and tested its effectiveness in mouse MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) model with the objectives to assess its potential use as an adjuvant therapy for PD. We used a subchronic MPTP model (5-daily MPTP injections), characterized by 50% loss of dopamine neurons over a period of 28 days. Ubisol-Q 10 was delivered in drinking water. Prophylactic application of Ubisol-Q 10 , started 2 weeks before the MPTP exposure, significantly offset the neurotoxicity (approximately 50% neurons died in MPTP group vs. 17% in MPTP+ Ubisol-Q 10 group by day 28). Therapeutic application of Ubisol-Q 10 , given after the last MPTP injection, was equally effective. At the time of intervention on day 5 nearly 25% of dopamine neurons were already lost, but the treatment saved the remaining 25% of cells, which otherwise would have died by day 28. This was confirmed by cell counts, analyses of striatal dopamine levels, and improved animals' motor skill on a beam walk test. Similar levels of neuroprotection were obtained with 3 different Ubisol-Q 10 concentrations tested, that is, 30 mg, 6 mg, or 3 mg CoQ 10 /kg body weight/day, showing clearly that high doses of CoQ 10 were not required to deliver these effects. Furthermore, the Ubisol-Q 10 treatments brought about a robust astrocytic activation in the brain parenchyma, indicating that astroglia played an active role in this neuroprotection. Thus, we have shown for the first time that Ubisol-Q 10 was capable of halting the neurodegeneration already in progress; *
Neutrophils represent a potential source of genotoxic reactive oxygen and nitrogen species in the... more Neutrophils represent a potential source of genotoxic reactive oxygen and nitrogen species in the tumor microenvironment. Using Mutatect cell lines, which can form subcutaneous tumors in syngeneic C57BL / 6 mice, we have previously established that the number of spontaneously infiltrating neutrophils correlates with the number of mutations at the hypoxanthine phosphoribosyltransferase (Hprt) locus. We now describe the properties of four lines that express different levels of the neutrophil chemokine, interleukin-8 (IL-8) , from a tetracycline (TET)-responsive promoter. In a series involving 45 animals, IL-8 ± expressing lines produced tumors with a higher neutrophil content than the control line. Analysis of the 45 tumors revealed that the neutrophil level again strongly correlated with hprt mutant frequency (MF) (P < .0001, r = 0.88). Administration of TET was effective in lowering the neutrophil content of low IL-8 ± expressing tumors, but not high IL-8 ± expressing tumors. Although the IL-8 transgene was stable in all lines in vitro, high IL-8 ± expressing lines completely lost the transgene in vivo whereas low IL-8 ± expressing lines showed no evidence of transgene instability. These results provide further evidence, based on the study of an endogenous gene (hprt) and an IL-8 transgene, that neutrophils may contribute to genetic instability in tumors. Neoplasia (2000) 2, 561 ± 568.
Mitochondrial cytopathies are associated with increased free radical generation and paracrystalli... more Mitochondrial cytopathies are associated with increased free radical generation and paracrystalline inclusions. Paracrystalline inclusions were serendipitously found in a young male athlete with a very high respiratory exchange ratio during steady-state exercise; he also had an unusually low aerobic capacity. Direct sequencing of the mitochondrial DNA (mtDNA) coding regions revealed a novel missense mutation (G15497A) resulting in a glycine3serine conversion at a highly conserved site in the cytochrome b gene in the subject, his mother, and sister. Cybrids, prepared by fusion of the subject's platelets with either U87MG °or SH-SY5Y °cells, generated higher basal levels of reactive oxygen species (ROS), had a lower adenosine triphosphate (ATP) content, and were more sensitive to oxygen and glucose deprivation and peroxynitrite generation compared to control cybrids with wild-type mtDNA. Cell survival was significantly enhanced with 50 mmol/L creatine monohydrate (CM) administration. The subject was also treated with CM (10 g/d) for a period of 5 weeks and a repeat muscle biopsy showed no paracrystalline inclusions. The results suggest that the development of exercise-induced paracrystalline inclusions may be influenced by the G15497A mtDNA mutation, and that CM mitigates against the pathological consequences of this mutation.
Astrocytes are the predominant cell type in the vicinity of glutamatergic synapses, where they mo... more Astrocytes are the predominant cell type in the vicinity of glutamatergic synapses, where they monitor and maintain low levels of glutamate. Synaptic homeostasis of glutamate involves its removal from the synaptic cleft via highaffinity glutamate transporters, glutamate transporter-1 (GLT-1)/excitatory amino acid transporters (EAAT)2 and glutamate and aspartate transporter (GLAST)/EAAT1, and glutamate-catabolizing enzyme, glutamine synthase. Glutamate transporters have been mostly characterized in rodent astrocytes, due to the lack of a convenient human cell system. We report here that NTera-2 (NT2/ D1, a cell line derived from a human teratocarcinoma and known to differentiate into neurons) can also be differentiated by a 4-week treatment with retinoic acid into functional astrocytes (NT2/A). Differentiation was accompanied by decreased cell proliferation and cell-cycle arrest, as measured by flow cytometry, immunostaining for Ki67 and incorporation of 5-bromo-2Јdeoxyuridine (BrdU). Immunocytochemistry and Western blot analysis showed that NT2/A expressed glial fibrillary acidic protein, vimentin and S100. Reverse transcription polymerase chain reaction (PCR) detected mRNA encoding glutamate transporters GLT-1/EAAT2 and GLAST/EAAT1. The expression level of GLAST/EAAT1 was higher than that of GLT-1/EAAT2, which is a typical expression pattern for primary astrocytes. Functionality of the transporters was demonstrated by the uptake of 3 H-glutamate. NT2/A also expressed active glutamine synthase, and treatment with glutamate (up to 1 mM for 24 hr) was non-toxic, suggesting that these cells were capable of converting it to non-toxic metabolites. NT2/A and NT2-derived neurons could be grown as mixed cultures and this may prove to be a useful experimental model to study molecular mechanisms underlying glutamate excitotoxicity.
Background: Vitamin E, an antioxidant, has been investigated for its effect on cancer incidence i... more Background: Vitamin E, an antioxidant, has been investigated for its effect on cancer incidence in humans, but no firm conclusions about a protective effect can be drawn from these studies. Recently, we reported a statistically significant correlation in the Mutatect mouse tumor model between the number of neutrophils and the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus. We have now used this model to investigate vitamin E's effect on the hprt mutation rate. Methods: Mutatect cells were grown in mice as subcutaneous tumors for 2-3 weeks, the tumor cells were recovered, and 6-thioguanine-resistant (i.e., hprt mutant) colonies were scored. Myeloperoxidase activity was used as a measure of neutrophil infiltration. Vitamin E (2 IU/kg body weight) was provided in the diet for 3-4 weeks. In some experiments, glyceryl trinitrate (100 mg/kg body weight) was also administered as a source of nitric oxide. All statistical tests were two-sided. Results: Mouse tumors from the Mutatect MN-11 cell line exhibited a 3.2-fold higher median mutation frequency than the same cells in culture (P<.0001); vitamin E reduced this frequency by 24.9% (P = .01). Mutatect TM-28-derived tumors (which secrete interleukin 8) were heavily infiltrated with neutrophils and had a correspondingly high mutation frequency; in two separate experiments, vitamin E reduced the median mutation frequency by 68.9% (P = .0019) and 84.1% (P = .011) and myeloperoxidase levels by 75.3% (P = .0002) and 75.5% (P = .026), respectively. Glyceryl trinitrate increased the mutation frequency in MN-11 tumors, and vitamin E reduced the median frequency by 61.4% (P = .058). Conclusions: Dietary vitamin E afforded strong protection against both spontaneously arising and nitric oxideinduced mutations. Two separate pro
Journal of Cerebral Blood Flow & Metabolism, 2007
Synaptic pathology is observed during hypoxic events in the central nervous system in the form of... more Synaptic pathology is observed during hypoxic events in the central nervous system in the form of altered dendrite structure and conductance changes. These alterations are rapidly reversible, on the return of normoxia, but are thought to initiate subsequent neuronal cell death. To characterize the effects of hypoxia on regulators of synaptic stability, we examined the temporal expression of cell adhesion molecules (CAMs) in synaptosomes after transient middle cerebral artery occlusion (MCAO) in mice. We focused on events preceding the onset of ischemic neuronal cell death (< 48 h). Synaptosome preparations were enriched in synaptically localized proteins and were free of endoplasmic reticulum and nuclear contamination. Electron microscopy showed that the synaptosome preparation was enriched in spheres (≈650 nm in diameter) containing secretory vesicles and postsynaptic densities. Forebrain mRNA levels of synaptically located CAMs was unaffected at 3 h after MCAO. This is contrast...
National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliognphic S... more National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliognphic SeMces sentices bibliographiques The author has granted a non-L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Libracy of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seU reproduire, prêter, distribuer ou copies of this thesis in microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. nie author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantid extracts 60m it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author' s ou autrement reproduits sans son permission. autorisation. Macrophage phagocytosis of polyethylene prticulate in vitro Master o f Applied Science,
Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins call... more Caveolae are plasma membrane invaginations that are enriched in cholesterol-binding proteins called caveolins. The presence of caveolae and caveolins in mixed cultures of human neurons and glia has not been investigated. Here, we sought to determine the presence of caveolae and caveolins in human NTera-2 (NT2/D1) cells, differentiated with retinoic acid into neuron-like (NT2/N) and astrocyte-like (NT2/A) cells. We found that while caveolin-3 mRNA levels remained relatively constant, caveolin-1 and -2 levels were upregulated in NT2/A and downregulated in NT2/N. No caveolin-1 immunoreactivity was detected in NT2/N. Electron microscopy revealed numerous flask-shaped invaginations (~86–102 nm in diameter) in the plasma membrane of NT2/A and NT2/N cells, while only few were detected in NT2/D1 cells. Immunoelectron microscopy localized caveolin-1 gold particles in the flask-shaped structures on plasmalemma and cytoplasmic vesicles of NT2/A cells. Furthermore, NT2/A endocytosed Alexa 488 c...
The world continues a desperate search for therapies that could bring hope and relief to millions... more The world continues a desperate search for therapies that could bring hope and relief to millions suffering from progressive neurodegenerative diseases such as Alzheimer’s (AD) and Parkinson’s (PD). With oxidative stress thought to be a core stressor, interests have long been focused on applying redox therapies including coenzyme-Q10. Therapeutic use has failed to show efficacy in human clinical trials due to poor bioavailability of this lipophilic compound. A nanomicellar, water-dispersible formulation of coenzyme-Q10, Ubisol-Q10, has been developed by combining coenzyme-Q10 with an amphiphilic, self-emulsifying molecule of polyoxyethanyl α-tocopheryl sebacate (derivatized vitamin E). This discovery made possible, for the first time, a proper assessment of the true therapeutic value of coenzyme-Q10. Micromolar concentrations of Ubisol-Q10 show unprecedented neuroprotection against neurotoxin exposure in in vitro and in vivo models of neurodegeneration and was extremely effective wh...
The coronavirus disease 2019 (COVID-19), a serious respiratory illness caused by the severe acute... more The coronavirus disease 2019 (COVID-19), a serious respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (SARS–CoV-2), has emerged as a global pandemic. Canada reported its first case of COVID-19 on the 25th January 2020. By March 2020, the virus had spread within Canadian communities reaching the most frail and vulnerable elderly population in long-term care facilities. The majority of cases were reported in the provinces of Quebec, Ontario, Alberta, and British Columbia, and the highest mortality was seen among individuals aged 65 years or older. Canada has the highest prevalence and incidence rates of several chronic inflammatory diseases, such as multiple sclerosis, inflammatory bowel disease, and Parkinson’s disease. Many elderly Canadians also live with comorbid medical illnesses, such as hypertension, diabetes, cardiovascular disease, and chronic lung disease, and are more likely to suffer from severe COVID-19 with a poor prognosis. It is becoming ...
Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essen... more Receptor-mediated transcytosis (RMT) is a principal pathway for transport of macromolecules essential for brain function across the blood-brain barrier (BBB). Antibodies or peptide ligands which bind RMT receptors are often co-opted for brain delivery of biotherapeutics. A constitutively recycling transferrin receptor (TfR) is a prototype receptor utilized to shuttle therapeutic cargos across the BBB. Several other BBB-expressed receptors have been shown to mediate transcytosis of ligands including insulin receptor (INSR) and insulin-like growth factor-1 receptor (IGF1R), lipid transporters LRP1, LDLR, LRP8 and TMEM30A, solute carrier family transporter LAT1 (subunit CD98hc) and leptin receptor (LEPR). In this study, we analyzed expression patterns of genes encoding RMT receptors in isolated brain microvessels, the brain and peripheral organs of the mouse and the human using RNAseq approach. IGF1R, INSR and LRP8 were highly enriched in the mouse brain microvessels compared to periph...
The immature embryos of Gossypium arboreum cv. RG8 were co-bombarded with two plasmids H1Z and pB... more The immature embryos of Gossypium arboreum cv. RG8 were co-bombarded with two plasmids H1Z and pBI121 using Biolistic® PDS-1000/He Particle Delivery System. The two plasmids were coated onto tungsten particles in the ratio 1:1 at a concentration of 100ng/μl which was 10 times less than recommended in the standard protocol. A total of 1827 embryos were bombarded in 12 different experiments. The representative embryos from each bombardment experiment were analysed 48 hr post bombardment through beta glucuronidase assay called GUS assay and PCR. The histochemical results showed indigo colouration in the representative embryos from 11 experiments (91.6%). The gene specific PCR analysis on genomic DNA isolated 48 hr post bombardment from 12 representative bombarded embryos revealed 1764bp amplicon corresponding to β-1, 3-glucanase ORF in nine (75%) bombarded embryos. The results confirmed successful co-bombardment of two transgenes despite using ten times less concentration of plasmid DNA.
A major emphasis in breeding for iron toxicity tolerance in rice is to identify differences that ... more A major emphasis in breeding for iron toxicity tolerance in rice is to identify differences that are associated with resistance and harness them for genetic improvement. In this study, thirty accessions, including IRRI gene bank accessions, two varieties from Brazil, 8 cultivars from West Africa and 10 cultivars from Uganda were analyzed for sensitivity to iron toxicity, and genetic diversity using morphological and SSR markers. Two genotypes, IR61612-313-16-2-2-1 and Suakoko 8 showed significantly high resistance with an average score of ≤ 3.5 on 1-9 scale. The SRR markers were highly informative and showed mean polymorphism information content (pic) of 0.68. The PIC values revealed that RM10793, RM3412, RM333, RM562, RM13628, RM310, RM5749, and RM154 could be the best markers for genetic diversity estimation of these rice cultivars. Diversity at the gene level showed an average of 4.61 alleles ranging from 2 to 12 per locus. Mean gene diversity (H) value for all SSR loci for the 30 genotypes evaluated was 0.69 but was decreased to 0.53 when analysis was performed on Ugandan accessions. The low genetic diversity found among the Ugandan accessions is the evidence of a narrow genetic base, and such a scenario has a potential vulnerability for resistance break down. A low correlation was detected between the observed molecular and morphological datasets. This means that a combination of morphological traits and SSR analysis would be required when assessing genetic variation under iron toxic conditions, and could be a practical strategy for breeders when planning crosses. A distinction between the resistant and susceptible accessions in both phenotyping and SSR datasets suggests the presence of unique alleles that could be harnessed for improvement of rice against iron toxicity.
Because nitric oxide related species have been found in the inflamed joints of patients with arth... more Because nitric oxide related species have been found in the inflamed joints of patients with arthritis, we investigated whether protein nitrotyrosine (a marker of tissue exposure to peroxynitrite) is present in their synovial tissues. Protein nitrotyrosine was detected immunohistochemically and by Western blot analysis. Synovial tissues removed surgically from 12 patients with rheumatoid arthritis (RA) (mean age 63.7 yrs) and 20 with osteoarthritis (OA) (mean age 66.6 yrs) were studied. Nitrated proteins were detected immunohistochemically in all of 18 tissues examined. Diffuse staining of the stroma was seen in all patients, with more extensive staining in RA than OA (p = 0.008). Intense staining was detected in some lymphocytes, but not in others, even within a single lymphoid aggregate. Neutrophils did not stain for nitrotyrosine. Vascular endothelial cells stained for nitrotyrosine but adjoining smooth muscle cells did not. Both cytoplasmic and nuclear staining was seen in macro...
The role of an A/T-rich positive regulatory region (P268, −444 to −177 from the translation start... more The role of an A/T-rich positive regulatory region (P268, −444 to −177 from the translation start site) of the pea plastocyanin gene (PetE) promoter has been investigated in transgenic plants containing chimeric promoters fused to the β-glucuronidase (GUS) reporter gene. This region enhanced GUS expression in leaves of transgenic tobacco plants when fused in either orientation to a minimal pea PetE promoter (−176 to +4) and in roots when fused in either orientation upstream or downstream of a minimal cauliflower mosaic virus 35S promoter (−90 to +5). The region was also able to enhance GUS expression in microtubers of transgenic potato plants when placed in either orientation upstream of a minimal class I patatin promoter (−332 to +14). Dissection of P268 revealed that cis elements responsible for enhancing GUS expression from the minimal PetE promoter were distributed throughout P268.
Mutation Research/Fundamental and Molecular Mechanisms of Mutagenesis, 1999
A new mouse model (Mutatect) that permits detection of mutations at the hprt (hypoxanthine phosph... more A new mouse model (Mutatect) that permits detection of mutations at the hprt (hypoxanthine phosphoribosyltransferase) locus is described. It is highly sensitive to detection of mutants induced by clastogenic agents such as ionizing radiation. MN-11 cells are grown as a subcutaneous tumour in C57BL/6 mice for a period of 2 weeks, during which time they can be exposed to mutagenic treatments. Cells taken from the animal are cultured ex vivo and 6-thioguanine (6-TG)-resistant mutant clones can be readily identified and scored. This model system may have special utility for detecting multi-locus deletion events (chromosomal mutations) induced by high LET forms of radiation that might be encountered in space.
The potential pathogenicity of two homoplasmic mtDNA point mutations, 9035TϾC and 4452TϾC, found ... more The potential pathogenicity of two homoplasmic mtDNA point mutations, 9035TϾC and 4452TϾC, found in a family afflicted with maternally transmitted cognitive developmental delay, learning disability, and progressive ataxia was evaluated using transmitochondrial cybrids. We confirmed that the 4452TϾC transition in tRNA Met represented a polymorphism; however, 9035TϾC conversion in the ATP6 gene was responsible for a defective F 0-ATPase. Accordingly, mutant cybrids had a reduced oligomycin-sensitive ATP hydrolyzing activity. They had less than half of the steady-state content of ATP and nearly an 8-fold higher basal level of reactive oxygen species (ROS). Mutant cybrids were unable to cope with additional insults, i.e., glucose deprivation or tertiary-butyl hydroperoxide, and they succumbed to either apoptotic or necrotic cell death. Both of these outcomes were prevented by the antioxidants CoQ 10 and vitamin E, suggesting that the abnormally high levels of ROS were the triggers of cell death. In conclusion, the principal metabolic defects, i.e., energy deficiency and ROS burden, resulted from the 9035TϾC mutation and could be responsible for the development of clinical symptoms in this family. Furthermore, antioxidant therapy might prove helpful in the management of this disease.
Parkinson's disease arises from a combination of environmental and genetic risk factors. At p... more Parkinson's disease arises from a combination of environmental and genetic risk factors. At present neither the curative nor preventative therapies are available; hence, there is an urgent need to develop reliable animal models to facilitate their development. Water soluble nanomiceller formulation of CoQ10 (Ubisol-Q10) has shown neuroprotection against neurotoxin on human neuronal cells. We have combined the genetic deficiency of DJ-1/PARK7 mice with MPTP exposure and develop a genetic susceptibility model of PD and evaluated the neuroprotective efficacy of (Ubisol-Q10). Transgenic mice with DJ-1 deficiency (DJ-1/PARK7) were given either water or Ubisol-Q10 prophylactically at a dose of 6 mg/kg/day added directly to a drinking water for one month followed challenged with MPTP injections while keeping the same drinking water regiments. Four weeks after the last injection we evaluated neuroprotective efficacy of Ubisol-Q10 in DJ-1/MPTP model of PD using histochemical and behavior...
Expression of the human respiratory syncytial virus (RSV) fusion protein (F) gene under the contr... more Expression of the human respiratory syncytial virus (RSV) fusion protein (F) gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter was analyzed by enzyme-linked immunosorbent assay (ELISA) in polyethylene glycol-transfected apple leaf protoplasts. In particular, we examined whether RSV-F gene expression could be enhanced by addition of a viral leader and a plant enhancer to the chimeric gene construct. Insertion of the 5′-untranslated leader from alfalfa mosaic virus (AMV) RNA 4 between the CaMV 35S promoter and the RSV-F gene increased viral expression by 5.5-fold compared to the construct without the leader. The addition of a transcriptional enhancer from the pea plastocyanin gene (PetE) upstream of the CaMV 35S promoter to a construct containing the AMV leader further increased RSV-F gene expression by 1.4-fold. Immunoblot assays showed that the RSV-F expressed in transfected apple protoplasts reacted with RSV-F monoclonal antibodies and was of the expected molecular mass of 68 kDa. These results demonstrated that the RSV-F recombinant protein was expressed in an antigenic form in plant cells. Furthermore, protein expression was enhanced by modifying the transfection vector using both a leader and an enhancer linked to a promoter.
Although the support for the use of antioxidants, such as coenzyme Q 10 (CoQ 10), to treat Parkin... more Although the support for the use of antioxidants, such as coenzyme Q 10 (CoQ 10), to treat Parkinson's disease (PD) comes from the extensive scientific evidence, the results of conducted thus far clinical trials are inconclusive. It is assumed that the efficacy of CoQ 10 is hindered by insolubility, poor bioavailability, and lack of brain penetration. We have developed a nanomicellar formulation of CoQ 10 (Ubisol-Q 10) with improved properties, including the brain penetration, and tested its effectiveness in mouse MPTP (1-methyl-4-phenyl-1, 2, 3, 6-tetrahydropyridine) model with the objectives to assess its potential use as an adjuvant therapy for PD. We used a subchronic MPTP model (5-daily MPTP injections), characterized by 50% loss of dopamine neurons over a period of 28 days. Ubisol-Q 10 was delivered in drinking water. Prophylactic application of Ubisol-Q 10 , started 2 weeks before the MPTP exposure, significantly offset the neurotoxicity (approximately 50% neurons died in MPTP group vs. 17% in MPTP+ Ubisol-Q 10 group by day 28). Therapeutic application of Ubisol-Q 10 , given after the last MPTP injection, was equally effective. At the time of intervention on day 5 nearly 25% of dopamine neurons were already lost, but the treatment saved the remaining 25% of cells, which otherwise would have died by day 28. This was confirmed by cell counts, analyses of striatal dopamine levels, and improved animals' motor skill on a beam walk test. Similar levels of neuroprotection were obtained with 3 different Ubisol-Q 10 concentrations tested, that is, 30 mg, 6 mg, or 3 mg CoQ 10 /kg body weight/day, showing clearly that high doses of CoQ 10 were not required to deliver these effects. Furthermore, the Ubisol-Q 10 treatments brought about a robust astrocytic activation in the brain parenchyma, indicating that astroglia played an active role in this neuroprotection. Thus, we have shown for the first time that Ubisol-Q 10 was capable of halting the neurodegeneration already in progress; *
Neutrophils represent a potential source of genotoxic reactive oxygen and nitrogen species in the... more Neutrophils represent a potential source of genotoxic reactive oxygen and nitrogen species in the tumor microenvironment. Using Mutatect cell lines, which can form subcutaneous tumors in syngeneic C57BL / 6 mice, we have previously established that the number of spontaneously infiltrating neutrophils correlates with the number of mutations at the hypoxanthine phosphoribosyltransferase (Hprt) locus. We now describe the properties of four lines that express different levels of the neutrophil chemokine, interleukin-8 (IL-8) , from a tetracycline (TET)-responsive promoter. In a series involving 45 animals, IL-8 ± expressing lines produced tumors with a higher neutrophil content than the control line. Analysis of the 45 tumors revealed that the neutrophil level again strongly correlated with hprt mutant frequency (MF) (P < .0001, r = 0.88). Administration of TET was effective in lowering the neutrophil content of low IL-8 ± expressing tumors, but not high IL-8 ± expressing tumors. Although the IL-8 transgene was stable in all lines in vitro, high IL-8 ± expressing lines completely lost the transgene in vivo whereas low IL-8 ± expressing lines showed no evidence of transgene instability. These results provide further evidence, based on the study of an endogenous gene (hprt) and an IL-8 transgene, that neutrophils may contribute to genetic instability in tumors. Neoplasia (2000) 2, 561 ± 568.
Mitochondrial cytopathies are associated with increased free radical generation and paracrystalli... more Mitochondrial cytopathies are associated with increased free radical generation and paracrystalline inclusions. Paracrystalline inclusions were serendipitously found in a young male athlete with a very high respiratory exchange ratio during steady-state exercise; he also had an unusually low aerobic capacity. Direct sequencing of the mitochondrial DNA (mtDNA) coding regions revealed a novel missense mutation (G15497A) resulting in a glycine3serine conversion at a highly conserved site in the cytochrome b gene in the subject, his mother, and sister. Cybrids, prepared by fusion of the subject's platelets with either U87MG °or SH-SY5Y °cells, generated higher basal levels of reactive oxygen species (ROS), had a lower adenosine triphosphate (ATP) content, and were more sensitive to oxygen and glucose deprivation and peroxynitrite generation compared to control cybrids with wild-type mtDNA. Cell survival was significantly enhanced with 50 mmol/L creatine monohydrate (CM) administration. The subject was also treated with CM (10 g/d) for a period of 5 weeks and a repeat muscle biopsy showed no paracrystalline inclusions. The results suggest that the development of exercise-induced paracrystalline inclusions may be influenced by the G15497A mtDNA mutation, and that CM mitigates against the pathological consequences of this mutation.
Astrocytes are the predominant cell type in the vicinity of glutamatergic synapses, where they mo... more Astrocytes are the predominant cell type in the vicinity of glutamatergic synapses, where they monitor and maintain low levels of glutamate. Synaptic homeostasis of glutamate involves its removal from the synaptic cleft via highaffinity glutamate transporters, glutamate transporter-1 (GLT-1)/excitatory amino acid transporters (EAAT)2 and glutamate and aspartate transporter (GLAST)/EAAT1, and glutamate-catabolizing enzyme, glutamine synthase. Glutamate transporters have been mostly characterized in rodent astrocytes, due to the lack of a convenient human cell system. We report here that NTera-2 (NT2/ D1, a cell line derived from a human teratocarcinoma and known to differentiate into neurons) can also be differentiated by a 4-week treatment with retinoic acid into functional astrocytes (NT2/A). Differentiation was accompanied by decreased cell proliferation and cell-cycle arrest, as measured by flow cytometry, immunostaining for Ki67 and incorporation of 5-bromo-2Јdeoxyuridine (BrdU). Immunocytochemistry and Western blot analysis showed that NT2/A expressed glial fibrillary acidic protein, vimentin and S100. Reverse transcription polymerase chain reaction (PCR) detected mRNA encoding glutamate transporters GLT-1/EAAT2 and GLAST/EAAT1. The expression level of GLAST/EAAT1 was higher than that of GLT-1/EAAT2, which is a typical expression pattern for primary astrocytes. Functionality of the transporters was demonstrated by the uptake of 3 H-glutamate. NT2/A also expressed active glutamine synthase, and treatment with glutamate (up to 1 mM for 24 hr) was non-toxic, suggesting that these cells were capable of converting it to non-toxic metabolites. NT2/A and NT2-derived neurons could be grown as mixed cultures and this may prove to be a useful experimental model to study molecular mechanisms underlying glutamate excitotoxicity.
Background: Vitamin E, an antioxidant, has been investigated for its effect on cancer incidence i... more Background: Vitamin E, an antioxidant, has been investigated for its effect on cancer incidence in humans, but no firm conclusions about a protective effect can be drawn from these studies. Recently, we reported a statistically significant correlation in the Mutatect mouse tumor model between the number of neutrophils and the frequency of mutation at the hypoxanthine phosphoribosyltransferase (hprt) locus. We have now used this model to investigate vitamin E's effect on the hprt mutation rate. Methods: Mutatect cells were grown in mice as subcutaneous tumors for 2-3 weeks, the tumor cells were recovered, and 6-thioguanine-resistant (i.e., hprt mutant) colonies were scored. Myeloperoxidase activity was used as a measure of neutrophil infiltration. Vitamin E (2 IU/kg body weight) was provided in the diet for 3-4 weeks. In some experiments, glyceryl trinitrate (100 mg/kg body weight) was also administered as a source of nitric oxide. All statistical tests were two-sided. Results: Mouse tumors from the Mutatect MN-11 cell line exhibited a 3.2-fold higher median mutation frequency than the same cells in culture (P<.0001); vitamin E reduced this frequency by 24.9% (P = .01). Mutatect TM-28-derived tumors (which secrete interleukin 8) were heavily infiltrated with neutrophils and had a correspondingly high mutation frequency; in two separate experiments, vitamin E reduced the median mutation frequency by 68.9% (P = .0019) and 84.1% (P = .011) and myeloperoxidase levels by 75.3% (P = .0002) and 75.5% (P = .026), respectively. Glyceryl trinitrate increased the mutation frequency in MN-11 tumors, and vitamin E reduced the median frequency by 61.4% (P = .058). Conclusions: Dietary vitamin E afforded strong protection against both spontaneously arising and nitric oxideinduced mutations. Two separate pro
Journal of Cerebral Blood Flow & Metabolism, 2007
Synaptic pathology is observed during hypoxic events in the central nervous system in the form of... more Synaptic pathology is observed during hypoxic events in the central nervous system in the form of altered dendrite structure and conductance changes. These alterations are rapidly reversible, on the return of normoxia, but are thought to initiate subsequent neuronal cell death. To characterize the effects of hypoxia on regulators of synaptic stability, we examined the temporal expression of cell adhesion molecules (CAMs) in synaptosomes after transient middle cerebral artery occlusion (MCAO) in mice. We focused on events preceding the onset of ischemic neuronal cell death (< 48 h). Synaptosome preparations were enriched in synaptically localized proteins and were free of endoplasmic reticulum and nuclear contamination. Electron microscopy showed that the synaptosome preparation was enriched in spheres (≈650 nm in diameter) containing secretory vesicles and postsynaptic densities. Forebrain mRNA levels of synaptically located CAMs was unaffected at 3 h after MCAO. This is contrast...
National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliognphic S... more National Library Bibliothèque nationale du Canada Acquisitions and Acquisitions et Bibliognphic SeMces sentices bibliographiques The author has granted a non-L'auteur a accordé une licence non exclusive licence allowing the exclusive permettant à la National Libracy of Canada to Bibliothèque nationale du Canada de reproduce, loan, distribute or seU reproduire, prêter, distribuer ou copies of this thesis in microfom, vendre des copies de cette thèse sous paper or electronic formats. la forme de microfiche/film, de reproduction sur papier ou sur format électronique. nie author retains ownership of the L'auteur conserve la propriété du copyright in this thesis. Neither the droit d'auteur qui protège cette thèse. thesis nor substantid extracts 60m it Ni la thèse ni des extraits substantiels may be printed or otherwise de celle-ci ne doivent être imprimés reproduced without the author' s ou autrement reproduits sans son permission. autorisation. Macrophage phagocytosis of polyethylene prticulate in vitro Master o f Applied Science,
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