My life as a dentist and ethicist: an experiment in creative nonfiction.

In a certain sense, we become the synthesis of our life experiences, and our professional identity is woven into a personal life story. A dentist with a PhD in Health Services Research and Policy who teaches clinical dentistry and ethics reflects on some of the moments that have combined to make her who she is.

BMCP1: a mitochondrial uncoupling protein in neurons which regulates mitochondrial function and oxidant production.

Outside the nervous system, members of the mitochondrial uncoupling protein (UCP) family have been proposed to contribute to control of body temperature and energy metabolism, and regulation of mitochondrial production of reactive oxygen species (ROS). However, the function of brain mitochondrial carrier protein 1 (BMCP1), which is highly expressed in brain, remains to be determined. To study BMCP1 expression and function in the nervous system, a high-affinity antibody to BMCP1 was generated and used to analyze tissue expression of BMCP1 protein in mouse. BMCP1 protein was highly expressed in heart and kidney, but not liver or lung. In the nervous system, BMCP1 was present in cortex, basal ganglia, substantia nigra, cerebellum, and spinal cord. Both BMCP1 mRNA and protein expression was almost exclusively neuronal. To study the effect of BMCP1 expression on mitochondrial function, neuronal (GT1-1) cell lines with stable overexpression of BMCP1 were generated. Transfected cells had higher State 4 respiration and lower mitochondrial membrane potential (psi(m)), consistent with greater mitochondrial uncoupling. BMCP1 expression also decreased mitochondrial production of ROS. These data suggest that BMCP1 can modify mitochondrial respiratory efficiency and mitochondrial oxidant production, and raise the possibility that BMCP1 might alter the vulnerability of brain to both acute injury and to neurodegenerative conditions.

Fullerene-based antioxidants and neurodegenerative disorders.

Water-soluble derivatives of buckminsterfullerene (C(60)) derivatives are a unique class of compounds with potent antioxidant properties. Studies on one class of these compounds, the malonic acid C(60) derivatives (carboxyfullerenes), indicated that they are capable of eliminating both superoxide anion and H(2)O(2), and were effective inhibitors of lipid peroxidation, as well. Carboxyfullerenes demonstrated robust neuroprotection against excitotoxic, apoptotic and metabolic insults in cortical cell cultures. They were also capable of rescuing mesencephalic dopaminergic neurons from both MPP(+) and 6-hydroxydopamine-induced degeneration. Although there is limited in vivo data on these compounds to date, we have previously reported that systemic administration of the C(3) carboxyfullerene isomer delayed motor deterioration and death in a mouse model of familial amyotrophic lateral sclerosis (FALS). Ongoing studies in other animal models of CNS disease states suggest that these novel antioxidants are potential neuroprotective agents for other neurodegenerative disorders, including Parkinson's disease.

Superoxide stress identifies neurons at risk in a model of ataxia-telangiectasia.

Ataxia-telangiectasia (A-T) is an autosomal recessive disorder caused by mutations in the ATM gene. A-T children demonstrate sensitivity to ionizing radiation, predisposition to hematological malignancies, and telangiectasias. However, the hallmark of A-T is fulminant degeneration of cerebellar Purkinje cells accompanied by a progressive ataxia with features of both cerebellar and basal ganglia dysfunction. Although the ATM gene product (ATM) is known to be involved in DNA repair, the mechanisms that link loss of ATM with neurodegeneration remain unknown. Recently, it has been suggested that abnormalities in redox status contribute to the A-T phenotype. To address this question in the nervous system, we measured reactive oxygen species (ROS) in brain regions and specific neuronal populations in ATM-/- mice. We found increased ROS levels in cerebellum and striatum but not cortex of ATM-/- mice compared to ATM+/+ mice. Confocal microscopic examination revealed elevated superoxide levels in cerebellar Purkinje cells and nigral dopaminergic neurons but not cortical neurons, thus mapping increased superoxide levels onto the neuronal populations selectively affected in A-T. These data are the first demonstration of elevated levels of ROS in neurons at risk in any genetic neurodegenerative disorder and, furthermore, suggest that ATM acts as a pro-survival signal in post-mitotic Purkinje cells and dopaminergic neurons by modifying superoxide radical handling in these selectively vulnerable neurons.

Clusterin contributes to caspase-3-independent brain injury following neonatal hypoxia-ischemia.

Clusterin, also known as apolipoprotein J, is a ubiquitously expressed molecule thought to influence a variety of processes including cell death. In the brain, it accumulates in dying neurons following seizures and hypoxic-ischemic (H-I) injury. Despite this, in vivo evidence that clusterin directly influences cell death is lacking. Following neonatal H-I brain injury in mice (a model of cerebral palsy), there was evidence of apoptotic changes (neuronal caspase-3 activation), as well as accumulation of clusterin in dying neurons. Clusterin-deficient mice had 50% less brain injury following neonatal H-I. Surprisingly, the absence of clusterin had no effect on caspase-3 activation, and clusterin accumulation and caspase-3 activation did not colocalize to the same cells. Studies with cultured cortical neurons demonstrated that exogenous purified astrocyte-secreted clusterin exacerbated oxygen/glucose-deprivation-induced necrotic death. These results indicate that clusterin may be a new therapeutic target to modulate non-caspase-dependent neuronal death following acute brain injury.

Rapid microplate assay for superoxide scavenging efficiency.

Here we report a method to determine superoxide scavenging efficiency, using kinetic analysis of cytochrome c reduction and an automated UV/vis microtiter plate reader. Superoxide (O(2)(-&z. rad;)) was generated by xanthine oxidase metabolism of hypoxanthine, and quantified by following reduction of cytochrome c by O(2)(-&z. rad;) as increasing absorbance at 550 nm. Reaction conditions were established that provided a linear increase in O(2)(-&z.rad;) generation for more than 20 min, and good reproducibility over time. The majority of cytochrome c reduction was blocked by superoxide dismutase, indicating cytochrome c reduction derived predominantly from O(2)(-&z.rad;). Although EDTA is commonly included in this assay to eliminate undesirable Fenton side-reactions with H(2)O(2) (a co-product of reactions that use xanthine oxidase to produce O(2)(-&z.rad;)), we found that catalase, but not EDTA, blocked suicide elimination of cytochrome c from the reaction. Finally, we demonstrate the feasibility of evaluating superoxide scavenging abilities on small samples extracted from two types of neuronal cultures, a hypothalamic neuronal cell line (GT1 trk cells) and primary mouse cortical cell cultures. This assay allows rapid, high throughput assessments of superoxide scavenging efficacy for small molecules of interest, as well as for cell or tissue extracts.

Localization of intra-abdominal thermoreceptors in the ewe.

1. In neutral environmental temperatures, shivering was induced in sheep by intra-ruminal cooling. Shivering was then depressed for a period by intra-abdominal heating. Intra-abdominal heating without ruminal cooling induced panting and a reduction of caval temperatures. Posterior caval temperatures were found to be inappropriate to the responses observed.2. Intra-abdominal heating of sheep in cold and warm environments depressed shivering and augmented panting respectively. Unilateral splanchnotomy abolished these responses on the ipsilateral side.3. The results are interpreted to indicate that the thermoreceptors stimulated lie within the walls of the rumen and intestine, and possibly the mesenteric veins.4. The splanchnic nerves are indicated as the afferent pathway for these receptors, and differential splanchnic innervation of the gut is suggested to explain unilateral abolition of the response to warming.

Thermoregulatory responses to intra-abdominal heating of sheep.

When electrical heat sources were implanted in the abdominal cavities of sheep and heated to dissipate 20 to 22 watts of additional endogenous heat in the animal, a rapid increase in respiratory frequency and respiratory water loss occurred 3 to 5 minutes after the initiation of heating. The response was accompanied by a marked decline of the temperature of the hypothalamus, with an increase of less than 1.0 degrees C in skin temperature over the location of the heaters in the abdomen. When the same skin area was heated externally in the absence of internal heating, no significant response was seen. The results support the concept of the existence of thermoreceptors, located in deep tissues or veins, which play a role in the regulation of body temperature.