Seizures are a common manifestation of hypoxic‐ischaemic brain injury in the neonate. In status epilepticus models alterations to GABAAR subunit expression have been suggested to contribute to (i) ...abnormal development of the GABAergic system, (ii) why seizures become self‐sustaining and (iii) the development of pharmacoresistance. Detailed investigation of GABAAR subunit protein expression after neonatal hypoxia‐ischaemia (HI) is currently insufficient. Using our pig model of HI and subsequent spontaneous neonatal seizures, we investigated changes in protein expression of the three predominant α‐subunits of the GABAAR; α1, α2 and α3. Anaesthetized, ventilated newborn pigs (< 24 h old) were subjected to 30 min HI and subsequently recovered to 24 or 72 h. Amplitude‐integrated electroencephalography was used to monitor brain activity and identify seizure activity. Brain tissue was collected post‐mortem and GABAAR α‐subunit protein expression was analysed using western blot and immunohistochemistry. GABAAR α1 and α3 protein expression was significantly reduced in animals that developed seizures after HI; HI animals that did not develop seizures did not exhibit the same reductions. Immunohistochemistry revealed decreased α1 and α3 expression, and α1 redistribution from the cell membrane to the cytosol, in the hippocampus of seizure animals. Multivariate analyses, controlling for HI severity and neuronal injury, revealed that seizures were independently associated with significant GABAAR α3 reduction. This is the first study to show loss and redistribution of GABAAR α‐subunits in a neonatal brain experiencing seizures. Our findings are similar to those reported in models of SE and in chronic epilepsy.
Treatments for seizures, phenobarbitone and diazepame, bind to the GABAA receptor. These are somewhat ineffective in the neonate. Using a piglet model of perinatal asphyxia, we investigated the expression of the major α‐subunits of the GABAAR. We observed decreased α1 and α3 expression, and seizures were independently associated with reduced α3 expression. Alterations to GABAAR‐subunit expression may explain the lack of treatment efficacy.
Glial fibrillary acidic protein (GFAP) is an enigmatic protein; it currently has no unambiguously defined role. It is expressed in the cytoskeleton of astrocytes in the mammalian brain. We have used ...co-immunoprecipitation to identify in vivo binding partners for GFAP in the rat and pig brain. We demonstrate interactions between GFAP, the glutamate transporter GLAST, the PDZ-binding protein NHERF1, and ezrin. These interactions are physiologically relevant; we demonstrate in vitro that transport of D-aspartate (a glutamate analogue) is significantly increased in the presence of GFAP and NHERF1. Moreover, we demonstrate in vivo that expression of GFAP is essential in retaining GLAST in the plasma membranes of astrocytes after an hypoxic insult. These data indicate that the cytoskeleton of the astrocyte plays an important role in protecting the brain against glutamate-mediated excitotoxicity.
In the gustatory system, the recognition of sugars, amino acids and bitter‐tasting compounds is the function of specialized G protein‐coupled receptors. Recently, two members of novel subfamily of G ...protein‐coupled receptors were proposed to function as taste receptors based on their specific expression in taste receptor cells. Here, we report the identification of a third member, T1R3, of this family of receptors. T1R3 maps near the telomere of mouse chromosome 4 rendering it a candidate for the Sac locus, a primary determinant of sweet preference in mice. Consistent with its candidacy for the Sac locus, T1R3 displays taste receptor cell‐specific expression. In addition, taster and non‐taster strains of mouse harbor different alleles of T1R3.
Recent studies, both in vitro and in vivo, have suggested the involvement of the polycystic kidney disease-1 and -2 like genes, Pkd1l3 and Pkd2l1, in acid taste transduction. In mice, disruption of ...taste cells expressing PKD2L1 eliminates gustatory neural responses to acids. However, no previous data exist on taste responses in the absence of PKD1L3 or on behavioral responses in mice lacking either of these proteins. In order to assess the function of PKD1L3, we genetically engineered mice with a targeted mutation of the Pkd1l3 gene. We then examined taste responsiveness of mutant and wild-type mice using several different approaches. In separate groups of mice, we measured preference scores in 48-h 2-bottle tests, determined NaCl or citric acid taste thresholds using a conditioned taste aversion technique, and conducted electrophysiological recordings of activity in the chorda tympani and glossopharyngeal nerves. Multiple taste compounds representing all major taste qualities were used in the preference tests and nerve-recording experiments. We found no significant reduction in taste responsiveness in Pkd1l3 mutant mice in behavioral or electrophysiological tests when compared with wild-type controls. Therefore, further studies are needed to elucidate the function of PKD1L3 in taste bud cells.
Glial fibrillary acidic protein (GFAP) is an intermediate filament protein expressed in the astrocyte cytoskeleton that plays an important role in the structure and function of the cell. GFAP can be ...phosphorylated at six serine (Ser) or threonine (Thr) residues but little is known about the role of GFAP phosphorylation in physiological and pathophysiological states. We have generated antibodies against two phosphorylated GFAP (pGFAP) proteins: p8GFAP, where GFAP is phosphorylated at Ser-8 and p13GFAP, where GFAP is phosphorylated at Ser-13. We examined p8GFAP and p13GFAP expression in the control neonatal pig brain and at 24 and 72 h after an hypoxic-ischemic (HI) insult. Immunohistochemistry demonstrated pGFAP expression in astrocytes with an atypical cytoskeletal morphology, even in control brains. Semi-quantitative western blotting revealed that p8GFAP expression was significantly increased at 24 h post-insult in HI animals with seizures in frontal, parietal, temporal and occipital cortices. At 72 h post-insult, p8GFAP and p13GFAP expression were significantly increased in HI animals with seizures in brain regions that are vulnerable to cellular damage (cortex and basal ganglia), but no changes were observed in brain regions that are relatively spared following an HI insult (brain stem and cerebellum). Increased pGFAP expression was associated with poor neurological outcomes such as abnormal encephalography and neurobehaviour, and increased histological brain damage. Phosphorylation of GFAP may play an important role in astrocyte remodelling during development and disease and could potentially contribute to the plasticity of the central nervous system.
The serum 25-hydroxyvitamin D 25(OH)D response to daily supplementation with 20 μg cholecalciferol (D3) during winter in predominantly white premenopausal women living in Maine was measured and the ...effects of body composition and hormonal contraceptive use on baseline serum 25(OH)D concentrations and the response to supplementation were examined. A total of 112 women (22.2 ± 3.7 y old) received placebo from March 2005 until September 2005 when they were randomized to receive either placebo or 20 μg/d D3 through February 2006. Eighty-six women completed the study. Actual mean D3 content of the supplements was 22 μg per capsule. In February 2005 the serum 25(OH)D concentration was 62.0 ± 23.4 nmol/L (mean ± SD). Serum 25(OH)D concentrations increased by 35.3 ± 23.2 nmol/L from February 2005 to February 2006 in the treatment group, significantly more than the 10.9 ± 16.9 nmol/L increase in the placebo group. Treatment group, magnitude of summer increase in 25(OH)D, estrogen dose, and baseline serum 25(OH)D concentrations, but not body fat, were significant predictors of the 1-y change in 25(OH)D concentrations used to assess the magnitude of the response to supplementation. Daily supplementation with 20 μg D3 during winter achieved optimal 25(OH)D concentrations (greater-than-or-equal75 nmol/L) in 80% of participants, indicating that this dose is adequate to optimize vitamin D status in most young women in Maine.
CD8+ T cells are crucial for control of a number of medically important protozoan parasites, including Trypanosoma cruzi, the agent of human Chagas disease. Yet, in contrast to the wealth of ...information from viral and bacterial infections, little is known about the antigen specificity or the general development of effector and memory T-cell responses in hosts infected with protozoans. In this study we report on a wide-scale screen for the dominant parasite peptides recognized by CD8+ T cells in T. cruzi-infected mice and humans. This analysis demonstrates that in both hosts the CD8+ T-cell response is highly focused on epitopes encoded by members of the large trans-sialidase family of genes. Responses to a restricted set of immunodominant peptides were especially pronounced in T. cruzi-infected mice, with more than 30% of the CD8+ T-cell response at the peak of infection specific for two major groups of trans-sialidase peptides. Experimental models also demonstrated that the dominance patterns vary depending on the infective strain of T. cruzi, suggesting that immune evasion may be occurring at a population rather than single-parasite level.
Seizures are a common manifestation of hypoxic-ischaemic brain injury in the neonate. In status epilepticus models alterations to GABA
R subunit expression have been suggested to contribute to (i) ...abnormal development of the GABAergic system, (ii) why seizures become self-sustaining and (iii) the development of pharmacoresistance. Detailed investigation of GABA
R subunit protein expression after neonatal hypoxia-ischaemia (HI) is currently insufficient. Using our pig model of HI and subsequent spontaneous neonatal seizures, we investigated changes in protein expression of the three predominant α-subunits of the GABA
R; α
, α
and α
. Anaesthetized, ventilated newborn pigs (< 24 h old) were subjected to 30 min HI and subsequently recovered to 24 or 72 h. Amplitude-integrated electroencephalography was used to monitor brain activity and identify seizure activity. Brain tissue was collected post-mortem and GABA
R α-subunit protein expression was analysed using western blot and immunohistochemistry. GABA
R α
and α
protein expression was significantly reduced in animals that developed seizures after HI; HI animals that did not develop seizures did not exhibit the same reductions. Immunohistochemistry revealed decreased α
and α
expression, and α
redistribution from the cell membrane to the cytosol, in the hippocampus of seizure animals. Multivariate analyses, controlling for HI severity and neuronal injury, revealed that seizures were independently associated with significant GABA
R α
reduction. This is the first study to show loss and redistribution of GABA
R α-subunits in a neonatal brain experiencing seizures. Our findings are similar to those reported in models of SE and in chronic epilepsy.
Learning about climate change is tangible when it addresses impacts that can be observed close to home. In this program, sixty-four diverse middle and high school students produced videos about ...locally relevant climate change topics. Graduate and undergraduate students provided mentorship. The program engaged students in research and learning about climate change, and sparked their interest in science careers. Evaluation results showed that students were highly motivated by the experience, developed a genuine interest in their science topic, learned about the scientific process, and developed twenty-first century skills. The program provided a unique and authentic approach to science learning and communication.
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